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Sample records for parapoxvirus orf virus

  1. Genomes of the Parapoxviruses Orf Virus and Bovine Papular Stomatitis Virus

    PubMed Central

    Delhon, G.; Tulman, E. R.; Afonso, C. L.; Lu, Z.; de la Concha-Bermejillo, A.; Lehmkuhl, H. D.; Piccone, M. E.; Kutish, G. F.; Rock, D. L.

    2004-01-01

    Bovine papular stomatitis virus (BPSV) and orf virus (ORFV), members of the genus Parapoxvirus of the Poxviridae, are etiologic agents of worldwide diseases affecting cattle and small ruminants, respectively. Here we report the genomic sequences and comparative analysis of BPSV strain BV-AR02 and ORFV strains OV-SA00, isolated from a goat, and OV-IA82, isolated from a sheep. Parapoxvirus (PPV) BV-AR02, OV-SA00, and OV-IA82 genomes range in size from 134 to 139 kbp, with an average nucleotide composition of 64% G+C. BPSV and ORFV genomes contain 131 and 130 putative genes, respectively, and share colinearity over 127 genes, 88 of which are conserved in all characterized chordopoxviruses. BPSV and ORFV contain 15 and 16 open reading frames (ORFs), respectively, which lack similarity to other poxvirus or cellular proteins. All genes with putative roles in pathogenesis, including a vascular endothelial growth factor (VEGF)-like gene, are present in both viruses; however, BPSV contains two extra ankyrin repeat genes absent in ORFV. Interspecies sequence variability is observed in all functional classes of genes but is highest in putative virulence/host range genes, including genes unique to PPV. At the amino acid level, OV-SA00 is 94% identical to OV-IA82 and 71% identical to BV-AR02. Notably, ORFV 006/132, 103, 109, 110, and 116 genes (VEGF, homologues of vaccinia virus A26L, A33R, and A34R, and a novel PPV ORF) show an unusual degree of intraspecies variability. These genomic differences are consistent with the classification of BPSV and ORFV as two PPV species. Compared to other mammalian chordopoxviruses, PPV shares unique genomic features with molluscum contagiosum virus, including a G+C-rich nucleotide composition, three orthologous genes, and a paucity of nucleotide metabolism genes. Together, these data provide a comparative view of PPV genomics. PMID:14671098

  2. A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein.

    PubMed

    Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier; Rziha, Hanns-Joachim

    2013-02-01

    The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (10(7) PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals. PMID:23175365

  3. Generation and Selection of Orf Virus (ORFV) Recombinants.

    PubMed

    Rziha, Hanns-Joachim; Rohde, Jörg; Amann, Ralf

    2016-01-01

    Orf virus (ORFV) is an epitheliotropic poxvirus, which belongs to the genus Parapoxvirus. Among them the highly attenuated, apathogenic strain D1701-V is regarded as a promising candidate for novel virus vector vaccines. Our recent work demonstrated that those ORFV-based recombinants were able to induce protective, long-lasting immunity in various hosts that are non-permissive for ORFV. In this chapter we describe procedures for the generation, selection, propagation, and titration of ORFV recombinants as well as transgene detection by PCR or immunohistochemical staining.

  4. Molecular Genetic Analysis of Orf Virus: A Poxvirus That Has Adapted to Skin

    PubMed Central

    Fleming, Stephen B.; Wise, Lyn M.; Mercer, Andrew A.

    2015-01-01

    Orf virus is the type species of the Parapoxvirus genus of the family Poxviridae. It induces acute pustular skin lesions in sheep and goats and is transmissible to humans. The genome is G+C rich, 138 kbp and encodes 132 genes. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique factors that allow it to replicate in the highly specific immune environment of skin. Phylogenetic analysis suggests that both viral interleukin-10 and vascular endothelial growth factor genes have been “captured” from their host during the evolution of the parapoxviruses. Genes such as a chemokine binding protein and a protein that binds granulocyte-macrophage colony-stimulating factor and interleukin-2 appear to have evolved from a common poxvirus ancestral gene while three parapoxvirus nuclear factor (NF)-κB signalling pathway inhibitors have no homology to other known NF-κB inhibitors. A homologue of an anaphase-promoting complex subunit that is believed to manipulate the cell cycle and enhance viral DNA synthesis appears to be a specific adaptation for viral-replication in keratinocytes. The review focuses on the unique genes of orf virus, discusses their evolutionary origins and their role in allowing viral-replication in the skin epidermis. PMID:25807056

  5. Suppression of influenza virus infection by the orf virus isolated in Taiwan

    PubMed Central

    LIN, Fong-Yuan; TSENG, Yeu-Yang; CHAN, Kun-Wei; KUO, Shu-Ting; YANG, Cheng-Hsiung; WANG, Chi-Young; TAKASU, Masaki; HSU, Wei-Li; WONG, Min-Liang

    2015-01-01

    Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection. PMID:25855509

  6. Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus).

    PubMed

    Nollens, Hendrik H; Jacobson, Elliott R; Gulland, Frances M D; Beusse, Diedrich O; Bossart, Gregory D; Hernandez, Jorge A; Klein, Paul A; Condit, Richard C

    2006-01-01

    Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.

  7. Activities of acyclic nucleoside phosphonates against Orf virus in human and ovine cell monolayers and organotypic ovine raft cultures.

    PubMed

    Dal Pozzo, F; Andrei, G; Holy, A; Van Den Oord, J; Scagliarini, A; De Clercq, E; Snoeck, R

    2005-12-01

    Orf virus, a member of the Parapoxvirus genus, causes a contagious pustular dermatitis in sheep, goats, and humans. Previous studies have demonstrated the activity of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; cidofovir; Vistide) against orf virus in cell culture and humans. We have evaluated a broad range of acyclic nucleoside phosphonates (ANPs) against several orf virus strains in primary lamb keratinocytes (PLKs) and human embryonic lung (HEL) monolayers. HPMPC, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6- diaminopurine (HPMPDAP), and (R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) were three of the most active compounds that were subsequently tested in a virus yield assay with PLK and HEL cells by virus titration and DNA quantification. HPMPC, HPMPDAP, and HPMPO-DAPy were evaluated for their activities against orf virus replication in organotypic epithelial raft cultures from differentiated PLK cells. At the highest concentrations (50 and 20 microg/ml), full protection was provided by the three drugs, while at 5 microg/ml, only HPMPDAP and HPMPC offered partial protection. The activities of the three compounds in the raft culture system were confirmed by quantification of infectious virus and viral DNA. These findings provide a rationale for the use of HPMPC and other ANPs in the treatment of orf (contagious ecthyma) in humans and animals.

  8. Isolation and characterization of orf viruses from Korean black goats.

    PubMed

    Oem, Jae-Ku; Chung, Joon-Yee; Kim, Yong-Joo; Lee, Kyoung-Ki; Kim, Seong-Hee; Jung, Byeong-Yeal; Hyun, Bang-Hun

    2013-01-01

    Five cases of orf virus infection in Korean black goats were diagnosed in our laboratory between 2010 and 2011. One orf virus (ORF/2011) was isolated from an ovine testis cell line (OA3.Ts) for use as a vaccine candidate. Sequences of the major envelope protein and orf virus interferon resistance genes were determined and compared with published reference sequences. Phylogenetic analyses revealed that orf viruses from Korean black goats were most closely related to an isolate (ORF/09/Korea) from dairy goats in Korea. This result indicates that the orf viruses might have been introduced from dairy goats into the Korean black goat population.

  9. Genome analysis of orf virus isolates from goats in the Fujian Province of southern China.

    PubMed

    Chi, Xuelin; Zeng, Xiancheng; Li, Wei; Hao, Wenbo; Li, Ming; Huang, Xiaohong; Huang, Yifan; Rock, Daniel L; Luo, Shuhong; Wang, Shihua

    2015-01-01

    Orf virus (ORFV), a species of the genus Parapoxvirus of the family Poxviridae, causes non-systemic, highly contagious, and eruptive disease in sheep, goat, and other wild and domestic ruminants. Our previous work shows orf to be ubiquitous in the Fujian Province of China, a region where there is considerable heterogeneity among ORFVs. In this study, we sequenced full genomes of four Fujian goat ORFV strains (OV-GO, OV-YX, OV-NP, and OV-SJ1). The four strains were 132-139 kb in length, with each containing 124-132 genes and about 64% G+C content. The most notable differences between the four strains were found near the genome termini. OV-NP lacked seven and OV-SJ1 lacked three genes near the right terminus when compared against other ORFVs. We also investigated the skin-virulence of the four Fujian ORFVs in goats. The ORFVs with gene deletions showed low virulence while the ORFVs without gene deletions showed high virulence in goats suggesting gene deletion possibly leads to attenuation of ORFVs. Gene 134 was disrupted in OV-NP genome due to the lack of initial code. The phylogenetic tree based on complete Parapoxviruse genomes showed that sheep originated and goat originated ORFVs formed distinctly separate branches with 100% bootstrap. Based on the single gene phylogenetic tree of 132 genes of ORFVs, 47 genes can be easily distinguished as having originated from sheep or goats. In order to further reveal genetic variation presented in goat ORFVs and sheep ORFVs, we analyzed the deduced amino acid sequences of gene 008, multiple alignment of amino acid sequences of gene 008 from the genome of five goat ORFVs and four sheep ORFVs revealed 33 unique amino acids differentiating it as having sheep or goats as host. The availability of genomic sequences of four Fujian goat ORFVs aids in our understanding of the diversity of orf virus isolates in this region and can assist in distinguishing between orf strains that originate in sheep and goats. PMID:26557108

  10. Looking back: a genetic retrospective study of Brazilian Orf virus isolates.

    PubMed

    Abrahão, J S; Borges, I A; Mazur, C; Lobato, Z I P; Ferreira, P C P; Bonjardim, C A; Trindade, G S; Kroon, E G

    2012-11-10

    Orf virus (ORFV), the prototype of the genus Parapoxvirus, is the aetiological agent of contagious ecthyma (CE), a pustular dermatitis that afflicts domestic and wild small ruminants. CE is one of the most widespread poxvirus diseases in the world, causing public health impacts. Outbreaks of ORFV have been observed in all geographical regions of Brazil, affecting ovine and caprine herds. The origins, epidemiology and identity of Brazilian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In the present study, we revisited CE outbreaks which occurred until 32 years ago, and we assessed, genetically, five viral isolates. We performed the sequencing and analysis of the three ORFV molecular markers: B2L gene, virus interferon resistance gene (VIR) and the vascular endothelial growth factor gene. Nucleotide and amino acid analysis of the analysed genes demonstrated that Brazilian ORFVs do not form a unique cluster, and presented more similarity to other worldwide ORFV samples than with each other. These data raise the questions of whether there are different worldwide ORFVs circulating in Brazil, or if all the Brazilian ORFV samples are of the same virus taken at distinct time points.

  11. Infection with Possible Novel Parapoxvirus in Horse, Finland, 2013.

    PubMed

    Airas, Niina; Hautaniemi, Maria; Syrjä, Pernilla; Knuuttila, Anna; Putkuri, Niina; Coulter, Lesley; McInnes, Colin J; Vapalahti, Olli; Huovilainen, Anita; Kinnunen, Paula M

    2016-07-01

    A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus. PMID:27315302

  12. Infection with Possible Novel Parapoxvirus in Horse, Finland, 2013

    PubMed Central

    Hautaniemi, Maria; Syrjä, Pernilla; Knuuttila, Anna; Putkuri, Niina; Coulter, Lesley; McInnes, Colin J.; Vapalahti, Olli; Huovilainen, Anita; Kinnunen, Paula M.

    2016-01-01

    A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus. PMID:27315302

  13. Comparative and retrospective molecular analysis of Parapoxvirus (PPV) isolates.

    PubMed

    Friederichs, Schirin; Krebs, Stefan; Blum, Helmut; Wolf, Eckhard; Lang, Heike; von Buttlar, Heiner; Büttner, Mathias

    2014-03-01

    Species members of the genus Parapoxvirus (PPV) within the family Poxviridae cause contagious pustular dermatitis in small ruminants (Orf virus, ORFV) and mostly mild localized inflammation in cattle (bovine papular stomatitis virus, BPSV and pseudocowpox virus, PCPV). All PPVs are known to be zoonotic, leading to circumscribed skin lesions in humans, historically known as milker's nodules. Human PPV isolates are often ill defined concerning their allocation to an animal origin. Here we present a comparative molecular analysis of a unique collection of 21 historic and recent human and animal PPV cell culture isolates (and two PPV DNA samples). Cell culture PPV propagation was restricted to primary ruminant fibroblasts and was strictly kept at low passages to avoid genomic changes by in vitro influences. For molecular arrangement of the isolate DNAs and their attribution to established PPV species DNA fragments of the PPVs were generated by two different discriminating PCR protocols, targeting the major part of the open reading frame (ORF) 011 (B2L gene) and the complete ORF 032. Multiple sequence alignments and phylogenetic analysis of both genes resulted in affiliation to the known PPV species. The sequences from the ORF 032 allowed discrimination of the isolate DNAs at a higher resolution. Human PPV isolates could be clearly assigned to the PPV species belonging to the reported or assumed animal host of transmission. For the first time, a whole PPV genome sequence comparison of a human biopsy derived virus (B029) and its ovine counterpart (B015) originating from a defined Orf outbreak in Germany is provided, revealing their well conserved relationship. Thus human PPVs can be molecularly retraced to the PPV species indicating the animal of transmission. After transmission to the human host, molecular conservation of the animal's virus peculiarities indicative for a PPV species became evident. PMID:24373950

  14. Experimental parapoxvirus infection (contagious ecthyma) in semi-domesticated reindeer (Rangifer tarandus tarandus).

    PubMed

    Tryland, Morten; Klein, Jörn; Berger, Therese; Josefsen, Terje D; das Neves, Carlos G; Oksanen, Antti; Åsbakk, Kjetil

    2013-03-23

    Contagious ecthyma (contagious pustular dermatitis, orf) occurs world-wide in sheep and goats and is caused by orf virus (genus Parapoxvirus, family Poxviridae). Contagious ecthyma outbreaks have been described in semi-domesticated reindeer (Rangifer tarandus tarandus) in Sweden, Finland and Norway, occasionally with high mortality. Fourteen one-year-old reindeer were corralled in mid-April. One week after arrival, two animals received a commercial live orf virus vaccine for sheep (Scabivax(®)) on scarified skin of the medial thigh. Four weeks later, the two vaccinated and six additional animals were inoculated in scarified oral mucosa with parapoxvirus obtained from reindeer with clinical contagious ecthyma. The remaining six reindeer were kept as sentinels, sharing feed and water with the inoculated animals. A small whitish lesion appeared on the inoculation site and the labial skin-mucosa junction of three animals five days post inoculation (p.i.). Twelve days p.i., typical ecthyma lesions were visible on the inoculation site in six of eight animals, including both vaccinees. Four inoculated animals (including both vaccinees) and one sentinel seroconverted 12 days p.i., and five animals (including one sentinel) seroconverted 20 days p.i. No contagious ecthyma-like lesions were detected in the sentinels. All animals were euthanized at 26-29 days p.i. Histological examination of lesions showed proliferative dermatitis with epidermal hyperplasia, hyperkeratosis, intra-epithelial pustules and ulcers. Orf virus DNA was detected in mandibular lymph nodes, tonsils and mucosal lesions of four animals, including one sentinel, which showed that virus transmission took place. The commercial orf virus vaccine may be difficult to administer due to the need for close-cropping and its zoonotic nature, and did not indicate significant protection, although the latter has to be verified with a larger number of animals.

  15. An Investigation of a Cluster of Parapoxvirus Cases in Missouri, Feb–May 2006: Epidemiologic, Clinical and Molecular Aspects

    PubMed Central

    Lederman, Edith R.; Tao, Min; Reynolds, Mary G.; Li, Yu; Zhao, Hui; Smith, Scott K.; Sitler, Lisa; Haberling, Dana L.; Davidson, Whitni; Hutson, Christina; Emerson, Ginny; Schnurr, David; Regnery, Russell; Zhu, Bao-Ping; Pue, Howard; Damon, Inger K.

    2013-01-01

    Simple Summary In the spring of 2006, four human cases of parapoxvirus infections in Missouri residents were reported to the Centers for Disease Control and Prevention (CDC). We conducted surveys of herders and veterinarians, performed animal and environmental sampling and obtained sera from potential case-patients. We determined that, in general, infected persons may seek advice from veterinarians rather than physicians, thereby giving physicians less clinical experience. The initial perception of increased incidence in Missouri was likely due to reporting bias due to misdiagnosis and increased awareness due to recent publications. Basic personal protective measures are not being routinely utilized. Asymptomatic parapoxvirus infections in livestock may be common and warrants further investigation. Abstract In the spring of 2006, four human cases of parapoxvirus infections in Missouri residents were reported to the Centers for Disease Control and Prevention (CDC), two of which were initially diagnosed as cutaneous anthrax. This investigation was conducted to determine the level of recognition of zoonotic parapoxvirus infections and prevention measures, the degree to which veterinarians may be consulted on human infections and what forces were behind this perceived increase in reported infections. Interviews were conducted and clinical and environmental sampling was performed. Swab and scab specimens were analyzed by real-time polymerase chain reaction (PCR), whereas serum specimens were evaluated for parapoxvirus antibodies. Three case patients were found to have fed ill juvenile animals without using gloves. Forty-six percent of veterinarians reported having been consulted regarding suspected human orf infections. Orf virus DNA was detected from five of 25 asymptomatic sheep. Analysis of extracellular envelope gene sequences indicated that sheep and goat isolates clustered in a species-preferential fashion. Parapoxvirus infections are common in Missouri ruminants

  16. Parapoxvirus (PPV) of red deer reveals subclinical infection and confirms a unique species.

    PubMed

    Friederichs, Schirin; Krebs, Stefan; Blum, Helmut; Lang, Heike; Büttner, Mathias

    2015-06-01

    Parapoxvirus (PPV) infections are of worldwide importance, particularly in sheep and goat herds. Owing to the zoonotic potential of all PPV species, they are a permanent threat to human health as well. The virus is also known to affect wildlife, as reported for pinnipeds, red deer and several other wild ruminants. PPVs found in red deer have been claimed as a unique species according to certain genomic features. So far infection of wildlife has been recognized because of clinical manifestation such as inflammation, stomatitis or typical pox-like lesions in the skin or mucous membranes. Here we report the use of targeted molecular diagnostics for the presence of PPV genomes in tonsil swabs of apparently healthy red deer in the Bavarian Alps. Out of 1764 swabs, 0.79 % tested positive for PPV genome presence. From one sample, PPV was successfully isolated in cell culture. This virus became the subject of complete genome characterization using next generation sequencing and various subsidiary PCR protocols. Strikingly, about a quarter of all ORFs were found to be larger than the corresponding ORFs in the reference PPV genome sequences used for comparison. To our knowledge this is the first genome-wide analysis that confirms red deer PPV as a unique species within the genus Parapoxvirus in Europe. Persistence of PPV in Alpine red deer indicates a source for virus transmission to susceptible livestock and hunters. The findings provide a further example of wildlife animals playing an important role as an inconspicuous reservoir of zoonotic diseases.

  17. Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins.

    PubMed

    Schoonbroodt, S; Piette, J; Baudoux, L; Defechereux, P; Rentier, B; Merville, M P

    1996-08-01

    Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle.

  18. Identification and Characterization of a Cleavage Site in the Proteolysis of Orf Virus 086 Protein

    PubMed Central

    Wang, Xiaoping; Xiao, Bin; Zhang, Jiafeng; Chen, Daxiang; Li, Wei; Li, Ming; Hao, Wenbo; Luo, Shuhong

    2016-01-01

    The orf virus (ORFV) is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus (VV) has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid) and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 h post infection at mRNA level and 12–24 h post infection at the protein level. The ORFV086 precursor and a 21 kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 h post infection, suggesting that both the ORFV086 precursor and the 21 kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 h post infection. The cleavage took place at different sites, resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21 kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21 kDa fragment post infection. Both ORFV086 precursor and the 21 kDa fragment are structural proteins of

  19. Human Orf virus infection from household exposures - United States, 2009-2011.

    PubMed

    2012-04-13

    Orf, also known as contagious ecthyma, is a zoonotic infection caused by a dermatotropic parapoxvirus that commonly infects sheep and goats; it is transmitted to humans through contact with an infected animal or fomites. In humans, orf manifests as an ulcerative skin lesion sometimes resembling bacterial infection or neoplasm. Human infection typically is associated with occupational animal contact and has been reported in children after visiting petting zoos and livestock fairs. Cases lacking these exposure histories might be misdiagnosed, leading to unnecessary treatment of orf lesions, which do not usually require any specific treatment. This report describes four cases of human orf associated with household meat processing or animal slaughter, highlighting the importance of nontraditional risk factors. Orf should be included in the differential diagnosis of patients with clinically compatible skin lesions and a history of household meat processing or animal slaughter. Persons and communities with these exposure risks also should receive counseling regarding the use of nonpermeable gloves and hand hygiene to prevent infection.

  20. Serological evidence for Parapoxvirus infection in chamois from the Tyrol regions of Austria and Italy.

    PubMed

    Huemer, Hartwig P; Zobl, Alexandra; Windisch, Andrea; Glawischnig, Walter; Büttner, Mathias; Kitchen, Maria; Trevisiol, Karin

    2014-01-01

    Orf-virus (ORFV) is a parapoxvirus that infects small ruminants worldwide causing sporadic zoonotic infections, mainly transmitted by direct contact with sheep and goats. Following an ORFV case in a hunter of Alpine chamois (Rupicapra rupicapra), who did not report previous contact to domestic animals, a serological survey in Western Austria was conducted to assess the seroprevalence of ORFV in this species. In addition, this study also tested blood/tissue samples of chamois from different areas of the adjacent province of Bolzano/Northern Italy for antibodies against ORFV using immunofluorescence and ELISA. The observed seropositivity rates in the chamois tested on the Austrian and Italian side of the Alps were 23.5% and 9.5%, respectively, with a combined 95% confidence interval ranging from 0.0678 to 0.238. Although the prevalence was significantly lower than the one observed in Austrian sheep flocks, this study provided the first evidence that parapoxviruses have spilled over into chamois populations to a significant degree in the Tyrol regions of Austria and Italy.

  1. Differential diagnosis of orf viruses by a single-step PCR.

    PubMed

    Chan, Kun-Wei; Hsu, Wei-Li; Wang, Chi-Young; Yang, Cheng-Hsiung; Lin, Fong-Yuan; Chulakasian, Songkhla; Wong, Min-Liang

    2009-09-01

    The complete nucleotide sequence of the A32L gene (named after vaccinia virus, corresponding with open reading frame 108 of the orf virus and encoding an ATPase) of the orf virus was studied using samples of orf virus from infected goats, which were collected from six outbreaks in central Taiwan. DNA sequence analysis of the A32L genes of these and isolates from other countries showed sequence heterogeneity (base pair variation and deletion) in the 3'-terminal regions. This finding led to the development of a polymerase chain reaction (PCR) method for the rapid differential diagnosis of orf virus infections, and the results demonstrated that this was an easy and reliable method for genotyping of orf viruses.

  2. Isolation and Characterization of Monoclonal Antibodies Against a Virion Core Protein of Orf Virus Strain NA1/11 As Potential Diagnostic Tool for Orf Viruses.

    PubMed

    Wang, Xiaoping; Zhang, Jiafeng; Hao, Wenbo; Peng, Yongzheng; Li, Hong; Li, Wei; Li, Ming; Luo, Shuhong

    2015-08-01

    Orf is caused by the orf virus (ORFV) and is a non-systemic, widespread disease afflicting sheep, goats, wild ruminants, and humans. Recent outbreaks in sheep and goats in Jilin and other northern Chinese provinces raise concerns about orf control in China. Thirty-five hybridoma clones were constructed from splenocytes of BALB/c mice immunized with natural orf virus protein. These hybridomas were used to produce antibodies targeting ORFV proteins. Immunological characterization of these monoclonal antibodies (MAb) showed that the 5F2D8 hybridoma line produced MAb that can recognize the 100, 70, and 20 kDa bands from total viral lysate. This hybridoma was further characterized by immunoprecipitation and peptide sequencing. The results indicate that 5F2D8 specifically recognizes orf virus encoded protein ORFV086, a late expression virion core protein that plays important roles in progeny virus particle assembly, morphogenesis, and maturity. Further experiments demonstrate that this MAb did not react with other viral proteins of ORFV orthopoxviruses, but reacted strongly to different field isolates of orf viruses from China. Additionally, this anti-ORFV086 MAb possesses ORFV neutralizing capability. Sequence alignments and phylogenetic analysis determined that ORFV086 of NA1/11, clustered together with NZ2 and IA82, is highly conserved and has structural similarities with the Vaccinia virus core protein P4a. As such, this MAb has great potential as a diagnostic tool for orf viruses, in the further exploration of orf pathogenesis, and in disease control and prevention.

  3. Orf Virus 002 Protein Targets Ovine Protein S100A4 and Inhibits NF-κB Signaling

    PubMed Central

    Chen, Daxiang; Zheng, Zewei; Xiao, Bin; Li, Wei; Long, Mingjian; Chen, Huiqin; Li, Ming; Rock, Daniel L.; Hao, Wenbo; Luo, Shuhong

    2016-01-01

    Orf virus (ORFV), a member of Parapoxvirus, has evolved various strategies to modulate the immune responses of host cells. The ORFV-encoded protein ORFV002, a regulator factor, has been found to inhibit the acetylation of NF-κB-p65 by blocking phosphorylation of NF-κB-p65 at Ser276 and also to disrupt the binding of NF-κB-p65 and p300. To explore the mechanism by which ORFV002 regulates NF-κB signaling, the understanding of ORFV002 potential binding partners in host cells is critical. In this study, ovine S100 calcium binding protein A4 (S100A4), prolyl endopeptidase-like (PREPL) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 8 (NDUFA8) were found to interact with ORFV002 based on the yeast two-hybrid (Y2H) assay using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). GST pull-down and bidirectional co-immunoprecipitation assay results demonstrate that ORFV002 interacts with S100A4 directly. Following the pEGFP-ORFV002 (p002GFP) transfection, we found that cytoplasmic S100A4 translocates into the nucleus and co-localizes with ORFV002. Furthermore, the inhibitory effect of ORFV002 on NF-κB signaling was significantly restored by S100A4 knock-down phenotype, suggesting that ovine S100A4 participates in the ORFV002-mediated NF-κB signaling. These data demonstrate that ORFV002 inhibits the NF-κB activation through its interaction with S100A4 along with its nucleus translocation.

  4. Orf Virus 002 Protein Targets Ovine Protein S100A4 and Inhibits NF-κB Signaling

    PubMed Central

    Chen, Daxiang; Zheng, Zewei; Xiao, Bin; Li, Wei; Long, Mingjian; Chen, Huiqin; Li, Ming; Rock, Daniel L.; Hao, Wenbo; Luo, Shuhong

    2016-01-01

    Orf virus (ORFV), a member of Parapoxvirus, has evolved various strategies to modulate the immune responses of host cells. The ORFV-encoded protein ORFV002, a regulator factor, has been found to inhibit the acetylation of NF-κB-p65 by blocking phosphorylation of NF-κB-p65 at Ser276 and also to disrupt the binding of NF-κB-p65 and p300. To explore the mechanism by which ORFV002 regulates NF-κB signaling, the understanding of ORFV002 potential binding partners in host cells is critical. In this study, ovine S100 calcium binding protein A4 (S100A4), prolyl endopeptidase-like (PREPL) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 8 (NDUFA8) were found to interact with ORFV002 based on the yeast two-hybrid (Y2H) assay using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). GST pull-down and bidirectional co-immunoprecipitation assay results demonstrate that ORFV002 interacts with S100A4 directly. Following the pEGFP-ORFV002 (p002GFP) transfection, we found that cytoplasmic S100A4 translocates into the nucleus and co-localizes with ORFV002. Furthermore, the inhibitory effect of ORFV002 on NF-κB signaling was significantly restored by S100A4 knock-down phenotype, suggesting that ovine S100A4 participates in the ORFV002-mediated NF-κB signaling. These data demonstrate that ORFV002 inhibits the NF-κB activation through its interaction with S100A4 along with its nucleus translocation. PMID:27679610

  5. Orf Virus 002 Protein Targets Ovine Protein S100A4 and Inhibits NF-κB Signaling.

    PubMed

    Chen, Daxiang; Zheng, Zewei; Xiao, Bin; Li, Wei; Long, Mingjian; Chen, Huiqin; Li, Ming; Rock, Daniel L; Hao, Wenbo; Luo, Shuhong

    2016-01-01

    Orf virus (ORFV), a member of Parapoxvirus, has evolved various strategies to modulate the immune responses of host cells. The ORFV-encoded protein ORFV002, a regulator factor, has been found to inhibit the acetylation of NF-κB-p65 by blocking phosphorylation of NF-κB-p65 at Ser(276) and also to disrupt the binding of NF-κB-p65 and p300. To explore the mechanism by which ORFV002 regulates NF-κB signaling, the understanding of ORFV002 potential binding partners in host cells is critical. In this study, ovine S100 calcium binding protein A4 (S100A4), prolyl endopeptidase-like (PREPL) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 8 (NDUFA8) were found to interact with ORFV002 based on the yeast two-hybrid (Y2H) assay using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). GST pull-down and bidirectional co-immunoprecipitation assay results demonstrate that ORFV002 interacts with S100A4 directly. Following the pEGFP-ORFV002 (p002GFP) transfection, we found that cytoplasmic S100A4 translocates into the nucleus and co-localizes with ORFV002. Furthermore, the inhibitory effect of ORFV002 on NF-κB signaling was significantly restored by S100A4 knock-down phenotype, suggesting that ovine S100A4 participates in the ORFV002-mediated NF-κB signaling. These data demonstrate that ORFV002 inhibits the NF-κB activation through its interaction with S100A4 along with its nucleus translocation. PMID:27679610

  6. 40 CFR 174.513 - Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene); exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Potato Leaf Roll Virus Resistance Gene... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.513 Potato Leaf Roll... protectant Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene) in or on all...

  7. 40 CFR 174.513 - Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene); exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Potato Leaf Roll Virus Resistance Gene... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.513 Potato Leaf Roll... protectant Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene) in or on all...

  8. 40 CFR 174.513 - Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene); exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Potato Leaf Roll Virus Resistance Gene... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.513 Potato Leaf Roll... protectant Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene) in or on all...

  9. 40 CFR 174.513 - Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene); exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Potato Leaf Roll Virus Resistance Gene... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.513 Potato Leaf Roll... protectant Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene) in or on all...

  10. 40 CFR 174.513 - Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene); exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Potato Leaf Roll Virus Resistance Gene... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.513 Potato Leaf Roll... protectant Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene) in or on all...

  11. Seroepidemiology of parapoxvirus infections in captive and free-ranging California sea lions Zalophus californianus.

    PubMed

    Nollens, Hendrik H; Gulland, Frances M D; Hernandez, Jorge A; Condit, Richard C; Klein, Paul A; Walsh, Michael T; Jacobson, Elliott R

    2006-04-01

    Cutaneous nodular lesions caused by parapoxvirus infections are commonly observed in stranded pinnipeds following their arrival at rehabilitation facilities. An indirect enzyme-linked immunosorbent assay (ELISA) was developed and validated to determine exposure to parapoxviruses in California sea lions Zalophus californianus in captivity and in the wild. The diagnostic performance of this assay was evaluated using receiver-operating characteristic analysis. At a selected cut-off value, the calculated sensitivity was 100% (95% CI = 86 to 100%) and the specificity was 100% (95% CI = 87 to 100%). Analysis of sera collected from 26 affected sea lions during various stages of the disease revealed anti-parapoxvirus antibodies in all affected sea lions prior to the development of cutaneous pox lesions. This indicated that previous exposure to a parapoxvirus does not confer protection against clinical disease. In at least 7 cases, exposure to the virus occurred during hospitalization. Analysis of paired sera from 74 unaffected sea lions indicated subclinical infections in at least 3 animals. Finally, the prevalence of anti-parapoxviral antibodies in 761 free-ranging California sea lions captured and tested was 91% (95% CI = 89 to 93%). This indicated that infection with a parapoxvirus is a common occurrence in the wild and that the release of captive sea lions infected with parapoxvirus into the wild should not increase the risk of a parapoxvirus outbreak in free-ranging sea lions. PMID:16724559

  12. Enzymatic Digestion Pattern of Varicella Zoster Virus ORF38 and ORF54 in Chickenpox Patients Using RFLP Technique

    PubMed Central

    Safarnezhad Tameshkel, Fahimeh; Karbalaie Niya, Mohammad Hadi; Keyvani, Hossein

    2016-01-01

    Background: Varicella zoster virus (VZV) causes chickenpox in children and zoster (zona) in the elderly. Using RFLP-PCR method for detection of VZV specific SNPs ORF38, 54 and 62 could distinguish the profile of VZV isolates. The aim of this study was to investigate enzymatic digestion pattern of VZV ORF38 and ORF54 in chickenpox patients using RFLP technique. Methods: Thirty-eight chickenpox patients, who referred to the hospitals of Iran University of Medical Sciences in Tehran from May 2010 to June 2015 were enrolled in this cross sectional study. After the DNA extraction, PCR amplification of 38 VZV isolates performed by specific primers of ORFs 38 and 54, then RFLP assay and digestion carried out by PstI (for ORF38) and BglI (for ORF54) restriction enzymes. Results: Of 38 positive VZV DNA, the mean age (yr)±SD was 34.4±23.3 (range: 7-89). 22 (57.9%) were female and 16 (42.1%) were male. The predominant VZV profile of BglI+PstI+ were 89.5% (34/38) followed by 10.5% (4/38) PstI+BglI‾. Statistical analysis showed that there was no significant relationship between genotype, age, sex, and year of infection variables (P value> 0.05). The common VZV genotype among Iranian patients with chickenpox and zona infection is genotype BglI+PstI+ followed by PstI+BglI‾. Conclusion: There are different VZV circulating genotypes that call for for more research on this field by widely population and other methods such as nucleotide sequencing to justify the accurate VZV genotype prevalence in Iran. PMID:26870141

  13. ORF9p phosphorylation by ORF47p is crucial for the formation and egress of varicella-zoster virus viral particles.

    PubMed

    Riva, Laura; Thiry, Marc; Bontems, Sebastien; Joris, Aline; Piette, Jacques; Lebrun, Marielle; Sadzot-Delvaux, Catherine

    2013-03-01

    The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

  14. Analysis of ORFs 2b, 3, 4, and partial ORF5 of sequential isolates of equine arteritis virus shows genetic variation following experimental infection of horses.

    PubMed

    Liu, Lihong; Castillo-Olivares, Javier; Davis-Poynter, Nick J; Baule, Claudia; Xia, Hongyan; Belák, Sándor

    2008-06-22

    Samples from horses experimentally infected with the "large plaque variant (LP3A+)" of equine arteritis virus were analysed. These included 182 nasal swabs collected from day 1 to 14 post-infection (p.i.), and 21 virus isolates obtained from white blood cells of animals that showed a prolonged viraemia between days 30 to 72 p.i. In order to determine the genetic stability of the virus and particularly to characterise the genetic variants found during the prolonged viraemia, partial sequences of open reading frame 5 (ORF5) encoding glycoprotein 5 (GP5) were generated. Viruses with amino acid substitutions in GP5 were used for further amplification and sequencing of a fragment encompassing ORFs 2b, 3, and 4. The ORF5 nucleotide sequences of the virus present in 65 out of 66 nasal swabs were identical to that of the inoculated virus, suggesting that the ORF5 gene of LP3A+ was genetically stable during the first 2 weeks p.i. Contrary to this, a number of mutations were found in the ORF5 of virus isolates obtained from day 30 p.i. The mutations mainly clustered in antigenic neutralization site C within variable region 1 of the GP5 ectodomain. Sequence variability was also identified in ORFs 2b, 3 and 4, with ORF 4 having the highest proportion of non-synonymous changes (4/6).

  15. Mapping regions of the cauliflower mosaic virus ORF III product required for infectivity.

    PubMed

    Jacquot, E; Geldreich, A; Keller, M; Yot, P

    1998-03-15

    The open reading frame (ORF) III product (PIII) of the pararetrovirus cauliflower mosaic virus (CaMV) has nucleic acid-binding properties in vitro, but its biological role is not yet determined. ORF III is closely linked to ORF II and overlaps ORF IV out of frame in the CaMV genome. A new CaMV-derived vector (Ca delta) devoid of ORF III and containing unique restriction sites between ORFs II and IV was designed. Introduction of the wild-type CaMV ORF III into Ca delta results in a clone (Ca3) infectious in turnip plants. Truncated or point-mutated versions of ORF III were then inserted into Ca delta and tested in vivo. Inoculation of the different mutants into turnip revealed that the four C-terminal amino acid residues of PIII are dispensable for infectivity as well as an internal domain (amino acids 61 to 80). Taken together the results show that PIII possesses a functional two-domain organization. Moreover, the CaMV PIII function(s) cannot be replaced either by the PIII protein of another caulimovirus, the figwort mosaic virus, or by the P2 protein of the cacao swollen shoot badnavirus, a member of the second plant pararetrovirus group.

  16. ORF virus infection in children: clinical characteristics, transmission, diagnostic methods, and future therapeutics.

    PubMed

    Lederman, Edith R; Austin, Connie; Trevino, Ingrid; Reynolds, Mary G; Swanson, Holly; Cherry, Bryan; Ragsdale, Jennifer; Dunn, John; Meidl, Susan; Zhao, Hui; Li, Yu; Pue, Howard; Damon, Inger K

    2007-08-01

    Orf virus leads to self-limited, subacute cutaneous infections in children who have occupational or recreational contact with infected small ruminants. Breaches in the integument and contact with animals recently vaccinated for orf may be important risk factors in transmission. Common childhood behaviors are likely important factors in the provocation of significant contact (ie, bites) or in unusual lesion location (eg, facial lesions). Clinician recognition is important in distinguishing orf infection from life-threatening cutaneous zoonoses. Recently developed molecular techniques provide diagnostic precision and newer topical therapeutics may hasten healing.

  17. Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability.

    PubMed

    Sun, Lichang; Zhou, Yan; Liu, Runxia; Li, Yanhua; Gao, Fei; Wang, Xiaomin; Fan, Hongjie; Yuan, Shishan; Wei, Zuzhang; Tong, Guangzhi

    2015-02-01

    ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.

  18. Identification of a hydrophobic domain in varicella-zoster virus ORF61 necessary for ORF61 self-interaction, viral replication, and skin pathogenesis.

    PubMed

    Wang, Li; Rajamani, Jaya; Sommer, Marvin; Zerboni, Leigh; Arvin, Ann M

    2013-04-01

    The varicella-zoster virus (VZV) ORF61 protein is necessary for normal replication in vitro and virulence in human skin xenografts in the severe combined immunodeficiency mouse model in vivo. These experiments identify a hydrophobic domain that mediates ORF61 self-interaction. While not needed to inhibit host cell defenses, disruption of this domain (residues 250 to 320) severely impairs VZV growth, transactivation of the immediate early 63 and glycoprotein E genes, and the pathogenesis of VZV skin infection in vivo.

  19. The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.

    PubMed Central

    Zafrullah, M; Ozdener, M H; Panda, S K; Jameel, S

    1997-01-01

    Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle. PMID:9371561

  20. Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels.

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Sivakumar, G; Tuteja, F C; Narnaware, S D; Mehta, S C; Singh, Raghvendar; Patil, N V

    2014-03-01

    The dsRNA binding protein (RBP) encoding gene of parapoxviruses (PPVs) from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV) from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV) and PCPV (reindeer PCPV and human PCPV) shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV) from different geographical areas of the world shared 69.5-71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV) in comparison to caprine and ovine PPV (ORFV). PMID:25685494

  1. Identification, phylogenetic evolutionary analysis of GDQY orf virus isolated from Qingyuan City, Guangdong Province, southern China.

    PubMed

    Duan, Chaohui; Liao, Meiying; Wang, Han; Luo, Xiaohong; Shao, Jing; Xu, Ying; Li, Wei; Hao, Wenbo; Luo, Shuhong

    2015-01-25

    Infection with the orf virus (ORFV) leads to contagious ecthyma, also called contagious pustular dermatitis, which usually affects sheep, goats and other small ruminants. It has a great distribution throughout the world and has also been reported to infect humans. Though many strains have been isolated from differing parts of mainland China, rarely has any strain been reported from the southern provinces of China. We studied a case of orf virus infection that occurred at Qingyuan City, Guangdong Province in southern China. An orf virus strain, GDQY, was successfully isolated and identified through cell culture techniques and transmission electron microscopy. Complete genes of ORFV011, ORFV059, ORFV106 and ORFV107 were amplified for the sequence analysis based on their nucleotide or amino acid level. In order to discuss the genetic variation, precise sequences were used to compare to other reference strains isolated from different districts or countries. Phylogenetic trees based on those strains were built up and evolutionary distances were calculated based on the alignment of their complete sequences. The typical structure of the orf virus was observed in cell-culture suspensions inoculated with GDQY, and the full-length of four genes was amplified and sequenced. Phylogenetic analysis indicated that GDQY is homologous to FJ-DS and CQ/WZ on ORFV011 nucleotides. ORFV059 may be more variable than ORFV011 based on the comparison between GDQY and other isolates. Genetic studies of ORFV106 and 107 are reported for the first time in the presented study.

  2. Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

    SciTech Connect

    Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana; Rozieres, Sohela de; Elder, John H.

    2008-02-20

    Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defective clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.

  3. Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus

    PubMed Central

    Tseng, Yeu-Yang; Liao, Guan-Ru; Sen, Ganes C.; Lin, Fong-Yuan

    2015-01-01

    ABSTRACT Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV. IMPORTANCE Our previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol 89:4966–4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20

  4. Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus.

    PubMed

    Tseng, Yeu-Yang; Liao, Guan-Ru; Sen, Ganes C; Lin, Fong-Yuan; Hsu, Wei-Li

    2015-11-01

    Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.IMPORTANCE Our previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol 89:4966-4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds

  5. Hepatitis E virus (HEV) protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.

    PubMed

    Paliwal, Daizy; Panda, Subrat Kumar; Kapur, Neeraj; Varma, Satya Pavan Kumar; Durgapal, Hemlata

    2014-08-01

    Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a non-enveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440-610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the ~35 kDa methyltransferase, ~35 kDa replicase and ~56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.

  6. Unique Presentation of Orf Virus Infection in a Thermal-Burn Patient After Receiving an Autologous Skin Graft.

    PubMed

    Hsu, Christopher H; Rokni, Ghasem Rahmatpour; Aghazadeh, Nessa; Brinster, Nooshin; Li, Yu; Muehlenbachs, Atis; Goldsmith, Cynthia S; Zhao, Hui; Petersen, Brett; McCollum, Andrea M; Reynolds, Mary G

    2016-10-15

    We describe a burn patient who developed skin lesions on her skin-graft harvest and skin-graft recipient (burn) sites. Orf virus infection was confirmed by a combination of diagnostic assays, including molecular tests, immunohistochemical analysis, pathologic analysis, and electron microscopy. DNA sequence analysis grouped this orf virus isolate among isolates from India. Although no definitive source of infection was determined from this case, this is the first reported case of orf virus infection in a skin graft harvest. Skin graft recipients with exposures to animals may be at risk for this viral infection. PMID:27456708

  7. ORF43 of Maize rayado fino virus is dispensable for systemic infection of maize and transmission by leafhoppers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize rayado fino virus (MRFV) possesses an open reading frame (ORF) encoding a protein with predicted mass of 43 kDa (ORF43) that has been postulated to be a viral movement protein. Using a clone of MRFV (pMRFV-US) from which infectious RNA can be produced, point mutations were introduced to eithe...

  8. Hepatitis E Virus Inhibits Type I Interferon Induction by ORF1 Products

    PubMed Central

    Nan, Yuchen; Yu, Ying; Ma, Zexu; Khattar, Sunil K.; Fredericksen, Brenda

    2014-01-01

    ABSTRACT Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-β) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. IMPORTANCE Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host

  9. Tiger frog virus ORF080L protein interacts with LITAF and impairs EGF-induced EGFR degradation.

    PubMed

    Chen, Yong-Shun; Chen, Nan-Nan; Qin, Xiao-Wei; Mi, Shu; He, Jian; Lin, Yi-Fan; Gao, Ming-Shi; Weng, Shao-Ping; Guo, Chang-Jun; He, Jian-Guo

    2016-06-01

    Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.

  10. Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells.

    PubMed

    Xu, Kailian; Guo, Shiyu; Zhao, Tianjing; Zhu, Huapei; Jiao, Hanwei; Shi, Qiaoyun; Pang, Feng; Li, Yaying; Li, Guohua; Peng, Dongmei; Nie, Xin; Cheng, Ying; Wu, Kebang; Du, Li; Cui, Ke; Zhang, Wenguang; Wang, Fengyang

    2016-01-01

    Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV. PMID:27648443

  11. Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells

    PubMed Central

    Guo, Shiyu; Zhao, Tianjing; Zhu, Huapei; Jiao, Hanwei; Shi, Qiaoyun; Pang, Feng; Li, Yaying; Li, Guohua; Peng, Dongmei; Nie, Xin; Wu, Kebang; Du, Li; Cui, Ke

    2016-01-01

    Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV.

  12. Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells

    PubMed Central

    Guo, Shiyu; Zhao, Tianjing; Zhu, Huapei; Jiao, Hanwei; Shi, Qiaoyun; Pang, Feng; Li, Yaying; Li, Guohua; Peng, Dongmei; Nie, Xin; Wu, Kebang; Du, Li; Cui, Ke

    2016-01-01

    Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV. PMID:27648443

  13. Varicella-zoster virus (VZV) mediates a delayed host shutoff independent of open reading frame (ORF) 17 expression.

    PubMed

    Waterboer, Tim; Rahaus, Markus; Wolff, Manfred H

    2002-01-01

    Varicella-zoster virus (VZV) open reading frame 17 (ORF 17) is the gene corresponding to Herpes simplex-virus (HSV) UL41. The UL41 gene encodes the virion host shutoff factor (vhs), a RNase that has been the object of detailed studies. In contrast to HSV, knowledge about VZV mediated shutoff effects and the role of ORF 17 is poor. We investigated the ORF 17 expression in infected cells and analyzed shutoff effects. ORF 17 expression could not be proven in infected human fibroblast cell lines and melanoma (MeWo) cells. Only after induction by Phorbol 12-myristate 13-acetate an ORF 17 expression became detectable in MeWo cells. Nevertheless, using stable expressed GAPDH mRNA as a marker for mRNA degradation, a VZV mediated shutoff, independent of ORF 17 expression, became measurable. Transfection experiments demonstrated that transient ORF 17 expression did not decrease the cellular GAPDH mRNA level. We examined whether the VZV shutoff factor is a tegument protein causing an early shutoff or whether it needs to be expressed (delayed shutoff). The GAPDH mRNA level in Actinomycin D pretreated and infected MeWo cells did not decrease even faster than the theoretical decay rate based on a half-life of 24 h. These findings lead to the conclusion that the VZV shutoff factor is not a mature protein localized in the virion and that VZV causes a delayed virion host shutoff effect.

  14. Role of autophagy in cellular response to infection with Orf virus Jilin isolate.

    PubMed

    Lan, Yungang; Wang, Gaili; Song, Deguang; He, Wenqi; Zhang, Di; Huang, Houshuang; Bi, Jingying; Gao, Feng; Zhao, Kui

    2016-09-25

    Autophagy is a conserved catabolic process of the cell, which has been described to be involved in the development of various viral diseases. However, the role of autophagy in Orf virus (ORFV) replication remains unknown. In this study, we provide the first evidence that ORFV infection triggered autophagy in primary ovine fetal turbinate cells (OFTu) based on the appearance of abundant double- and single-membrane vesicles, the accumulation of LC3 fluorescent puncta, the enhancement of LC3-I/-II conversion, and autophagic flux. Moreover, modulation of ORFV-induced autophagy by rapamycin (RAPA), Earle's balanced salts solution (EBSS), chloroquine (CQ) or 3-methyladenime (3-MA) does not affect virus production. In conclusion, these results suggest that autophagy can be induced in host cells by ORFV infection, but which maybe not essential for ORFV replication. PMID:27599926

  15. Intracellular localization of varicella-zoster virus ORF39 protein and its functional relationship to glycoprotein K

    SciTech Connect

    Govero, Jennifer; Hall, Susan; Heineman, Thomas C. . E-mail: heinemtc@slu.edu

    2007-02-20

    Varicella-zoster virus (VZV) encodes two multiply inserted membrane proteins, open reading frame (ORF) 39 protein (ORF39p) and glycoprotein K (gK). The HSV-1 homologs of these proteins are believed to act in conjunction with each other during viral egress and cell-cell fusion, and they directly influence each other's intracellular trafficking. However, ORF39p and VZV gK have received very limited study largely due to difficulties in producing antibodies to these highly hydrophobic proteins. To overcome this obstacle, we introduced epitope tags into both ORF39p and gK and examined their intracellular distributions in transfected and infected cells. Our data demonstrate that both ORF39p and gK accumulate predominately in the ER of cultured cells when expressed in the absence of other VZV proteins or when coexpressed in isolation from other VZV proteins. Therefore, the transport of VZV ORF39p and gK does not exhibit the functional interdependence seen in their HSV-1 homologs. However, during infection, the primary distributions of ORF39p and gK shift from the ER to the Golgi, and they are also found in the plasma membrane indicating that their intracellular trafficking during infection depends on other VZV-encoded proteins. During infection, ORF39p and gK tightly colocalize with VZV envelope glycoproteins B, E and H; however, the coexpression of ORF39p or gK with other individual viral glycoproteins is insufficient to alter the transport of either ORF39p or gK.

  16. Molecular detection and analysis of Sheeppox and Orf viruses isolated from sheep from Qalubia, Egypt.

    PubMed

    Selim, Abdelfattah; Elhaig, Mahmoud; Höche, Jennifer; Gaede, Wolfgang

    2016-01-01

    In this study an outbreak with Sheeppox virus (SPPV) and Orf virus (ORFV) in one sheep herd in the Qalubia province, Egypt, was investigated. Both, SPPV and ORFV caused clinically manifest infections among sheep. The affected sheep showed skin lesions around the mouth or all over the body. Therefore, reliable diagnosis should confirm the aetiology of the infection and then reduce spread of the diseases in the affected areas. Clinical samples were investigated by virus isolation, PCR and real-time PCR assays. Furthermore, PCR-products of SPPV and ORFV isolates were sequenced and alignment to reference isolates was performed for phylogenetic analyses. The laboratory diagnosis showed that real-time PCR assay was more accurate and sensitive than conventional PCR and virus isolation. In phylogenetic analysis of the A29L gene genetic differences between SPPV field strains were not observed and the strains showed 100% homology with two SPPV isolates from Kazakhstan and one isolate from Turkey. The ORFV field strains are in the P55 gene genetically distinct from another and from other published isolates from Egypt 2006 and 2009. PMID:27529993

  17. Development of a lateral flow immunochromatographic assay for the rapid diagnosis of Orf virus infections.

    PubMed

    Zhao, Kui; He, Wenqi; Bi, Jingying; Zhang, Ximu; Zhang, Di; Huang, Houshuang; Zhang, Yuexiang; Song, Deguang; Gao, Feng

    2016-10-01

    A rapid and simple lateral-flow immunochromatographic assay (LFIA) was developed for the specific detection of Orf virus (ORFV) using two distinct monoclonal antibodies (MAbs: 5A5 and 6F2) against the ORFV ORF011 protein. The MAb 5A5 was conjugated with colloidal gold, and the MAb 6F2 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated test (T) and control (C), respectively. The results showed that samples of ORFV complexed with colloidal gold-conjugated MAb 5A5, were captured by MAb 6F2 at the T line resulting in the appearance of a purple band. When samples did not contain ORFV or when they contained a quantity of ORFV below the detection limit of the test, only the C line was visible. The analysis of sensitivity of the test demonstrated that the lowest detected quantity of ORFV was 2.03×10(3.0) TCID50/ml. Storage at room temperature for 6 months did not result in the loss of performance of the LFIA test. Using loop-mediated isothermal amplification (LAMP) as a reference test, the relative specificity and sensitivity of the LFIA test were determined to be 100% and 92.1%, respectively. Based on these results, the LFIA test developed may be a suitable tool for rapid on-site testing for ORFV infection. PMID:27380632

  18. The ORF012 Gene of Marek's Disease Virus Type 1 Produces a Spliced Transcript and Encodes a Novel Nuclear Phosphoprotein Essential for Virus Growth

    PubMed Central

    Schippers, Timo; Jarosinski, Keith

    2014-01-01

    ABSTRACT Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV carries a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potentials and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique long region. Using reverse transcriptase PCR, we showed that ORF011 and ORF012 are not individual genes but form a single gene through mRNA splicing of a small intron, resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defect in vitro and could not be passaged in cultured cells, suggesting an essential role for the ORF012 product in virus replication. Further studies revealed that protein 012 (p012) localized to the nucleus in transfected and infected cells, and we identified by site-directed mutagenesis and green fluorescent protein (GFP) reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23-amino-acid sequence at the protein's C terminus. Nuclear export was blocked using leptomycin B, suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution. IMPORTANCE Marek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle, which eventually could help us to

  19. In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

    PubMed

    Wang, Gaili; He, Wenqi; Song, Deguang; Li, Jida; Bao, Yingfu; Lu, Rongguang; Bi, Jingying; Zhao, Kui; Gao, Feng

    2014-05-01

    Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

  20. Sequence diversity on four ORFs of citrus tristeza virus correlates with pathogenicity

    PubMed Central

    Herrera-Isidrón, Lisset; Ochoa-Sánchez, Juan Carlos; Rivera-Bustamante, Rafael; Martínez-Soriano, Juan Pablo

    2009-01-01

    The molecular characterization of isolates of citrus tristeza virus (CTV) from eight locations in Mexico was undertaken by analyzing five regions located at the opposite ends of the virus genome. Two regions have been previously used to study CTV variability (coat protein and p23), while the other three correspond to other genomic segments (p349-B, p349-C and p13). Our comparative nucleotide analyses included CTV sequences from different geographical origins already deposited in the GenBank databases. The largest nucleotide differences were located in two fragments located at the 5' end of the genome (p349-B and p349-C). Phylogenetic analyses on those five regions showed that the degree of nucleotide divergence among strains tended to correlate with their pathogenicity. Two main groups were defined: mild, with almost no noticeable effects on the indicator plants and severe, with drastic symptoms. Mild isolates clustered together in every analyzed ORF sharing a genetic distance below 0.022, in contrast with the severe isolates, which showed a more disperse distribution and a genetic distance of 0.276. Analyses of the p349-B and p349-C regions evidenced two lineages within the severe group: severe common subgroup (most of severe isolates) and severe divergent subgroup (T36-like isolates). This study represents the first attempt to analyze the genetic variability of CTV in Mexico by constructing phylogenetic trees based on new genomic regions that use group-specific nucleotide and amino acid sequences. These results may be useful to implement specific assays for strain discrimination. Moreover, it would be an excellent reference for the CTV situation in México to face the recent arrival of brown citrus aphid. PMID:19642988

  1. Purifying selection in Porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

    PubMed Central

    Robinson, Sally R.; Abrahante, Juan E.; Johnson, Craig R.; Murtaugh, Michael P.

    2013-01-01

    Porcine Reproductive and Respiratory Syndrome Virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3,466 Type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS. PMID:24084290

  2. Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector

    PubMed Central

    Farshadpour, Fatemeh; Makvandi, Manoochehr; Taherkhani, Reza

    2015-01-01

    Background: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. Objectives: The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. Materials and Methods: A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. Results: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations. PMID

  3. ORF7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against IFN-α-induced antiviral response.

    PubMed

    Dedeurwaerder, Annelike; Olyslaegers, Dominique A J; Desmarets, Lowiese M B; Roukaerts, Inge D M; Theuns, Sebastiaan; Nauwynck, Hans J

    2014-02-01

    The type I IFN-mediated immune response is the first line of antiviral defence. Coronaviruses, like many other viruses, have evolved mechanisms to evade this innate response, ensuring their survival. Several coronavirus accessory genes play a central role in these pathways, but for feline coronaviruses this has never to our knowledge been studied. As it has been demonstrated previously that ORF7 is essential for efficient replication in vitro and virulence in vivo of feline infectious peritonitis virus (FIPV), the role of this ORF in the evasion of the IFN-α antiviral response was investigated. Deletion of ORF7 from FIPV strain 79-1146 (FIPV-Δ7) rendered the virus more susceptible to IFN-α treatment. Given that ORF7 encodes two proteins, 7a and 7b, it was further explored which of these proteins is active in this mechanism. Providing 7a protein in trans rescued the mutant FIPV-Δ7 from IFN sensitivity, which was not achieved by addition of 7b protein. Nevertheless, addition of protein 7a to FIPV-Δ3Δ7, a FIPV mutant deleted in both ORF3 and ORF7, could no longer increase the replication capacity of this mutant in the presence of IFN. These results indicate that FIPV 7a protein is a type I IFN antagonist and protects the virus from the antiviral state induced by IFN, but it needs the presence of ORF3-encoded proteins to exert its antagonistic function.

  4. Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

    PubMed Central

    Tao, Xue Ying; Choi, Jae Young; Kim, Woo Jin; An, Saes Byeol; Liu, Qin; Kim, Song Eun; Lee, Seok Hee; Kim, Jong Hoon; Woo, Soo Dong; Jin, Byung Rae

    2014-01-01

    ABSTRACT ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5′ rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. IMPORTANCE Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles. PMID:25320313

  5. hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles

    PubMed Central

    Rodriguez-Frandsen, Ariel; de Lucas, Susana; Pérez-González, Alicia; Pérez-Cidoncha, Maite; Roldan-Gomendio, Alejandro; Pazo, Alejandra; Marcos-Villar, Laura; Landeras-Bueno, Sara; Ortín, Juan; Nieto, Amelia

    2016-01-01

    The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus. PMID:26864902

  6. Molecular characterization of the spike and ORF3 genes of porcine epidemic diarrhea virus in the Philippines.

    PubMed

    Kim, Yong Kwan; Cho, Yoon-Young; An, Byung-Hyun; Lim, Seong-In; Lim, Ji-Ae; Cho, In-Soo; Le, Van Phan; An, Dong-Jun

    2016-05-01

    Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. In the present study, we analyzed the spike genes and ORF3 genes of seven PEDV strains detected in Philippine pigs in June 2014. There are four major epitope regions in the spike glycoprotein: a CO-26K equivalent (COE) domain, SS2 and SS6 epitopes, and an epitope region recognized by the 2C10 monoclonal antibody. Analysis of Philippine strains revealed amino acid substitutions in the SS6 epitope region (LQDGQVKI to SQSGQVKI) of the S1 domain. Substitutions were also detected in the 2C10 epitope region (GPRLQPY to GPRFQPY) in the cytoplasmic domain. Phylogenetic analysis of the complete spike gene sequences from the seven strains revealed that they clustered within the G2 group but were distantly related to the North American and INDELs clusters. Interestingly, these strains were close to Vietnamese PEDVs on the ORF3 genetic tree and showed high (97.0-97.6 %) sequence identity to ORF3 genes at the nucleotide level.

  7. Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus.

    PubMed

    Martínez-Turiño, Sandra; Hernández, Carmen

    2010-12-01

    Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5'-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates. PMID:20826617

  8. Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus.

    PubMed

    Martínez-Turiño, Sandra; Hernández, Carmen

    2010-12-01

    Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5'-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.

  9. Inactivated ORF virus shows antifibrotic activity and inhibits human hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in preclinical models.

    PubMed

    Paulsen, Daniela; Urban, Andreas; Knorr, Andreas; Hirth-Dietrich, Claudia; Siegling, Angela; Volk, Hans-Dieter; Mercer, Andrew A; Limmer, Andreas; Schumak, Beatrix; Knolle, Percy; Ruebsamen-Schaeff, Helga; Weber, Olaf

    2013-01-01

    Inactivated orf virus (iORFV), strain D1701, is a potent immune modulator in various animal species. We recently demonstrated that iORFV induces strong antiviral activity in animal models of acute and chronic viral infections. In addition, we found D1701-mediated antifibrotic effects in different rat models of liver fibrosis. In the present study, we compare iORFV derived from two different strains of ORFV, D1701 and NZ2, respectively, with respect to their antifibrotic potential as well as their potential to induce an antiviral response controlling infections with the hepatotropic pathogens hepatitis C virus (HCV) and hepatitis B virus (HBV). Both strains of ORFV showed anti-viral activity against HCV in vitro and against HBV in a transgenic mouse model without signs of necro-inflammation in vivo. Our experiments suggest that the absence of liver damage is potentially mediated by iORFV-induced downregulation of antigen cross-presentation in liver sinus endothelial cells. Furthermore, both strains showed significant anti-fibrotic activity in rat models of liver fibrosis. iORFV strain NZ2 appeared more potent compared to strain D1701 with respect to both its antiviral and antifibrotic activity on the basis of dosages estimated by titration of active virus. These results show a potential therapeutic approach against two important human liver pathogens HBV and HCV that independently addresses concomitant liver fibrosis. Further studies are required to characterize the details of the mechanisms involved in this novel therapeutic principle.

  10. A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V105DLP108 Immunoactivity.

    PubMed

    Wang, Heng; Ji, Fangxiao; Liang, Huanbin; Gu, Honglang; Ning, Zhangyong; Liu, Rongchang; Zhang, Guihong

    2015-01-01

    The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region. PMID:26177202

  11. A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V105DLP108 Immunoactivity

    PubMed Central

    Gu, Honglang; Ning, Zhangyong; Liu, Rongchang; Zhang, Guihong

    2015-01-01

    The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region. PMID:26177202

  12. Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

    PubMed Central

    Fu, Bishi; Kuang, Ersheng; Li, Wenwei; Avey, Denis; Li, Xiaojuan; Turpin, Zachary; Valdes, Ahmed; Brulois, Kevin; Myoung, Jinjong

    2014-01-01

    ABSTRACT We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838–1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication. IMPORTANCE KSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK

  13. Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins

    PubMed Central

    Olsen, Christel M.; Markussen, Turhan; Thiede, Bernd; Rimstad, Espen

    2016-01-01

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus infecting salmonid fish. The virus is adapted to low temperature and has a replication optimum between 10–15 °C. In this study the subcellular localization and protein interactions for the protein encoded by the largest open reading frame of gene segment 8 (s8ORF2) were investigated. In ISAV infected cells the s8ORF2 protein was found mainly in the cytosol but a minor fraction of cells expressed the protein in the nucleus as well. Green fluorescent protein-tagged s8ORF2 did not leak out of the cell when the plasma membrane was permeabilized, suggesting interactions with intracellular structural components. The s8ORF2 protein exists both as monomer and homodimer, and co-immunoprecipitation experiments strongly suggests it binds to the ISAV fusion-, nucleo- and matrix proteins. Two versions of s8ORF2 were detected with apparent molecular weights of 24–26 and 35 kDa in lysates of infected cells. The 35 kDa type is an early viral protein while the smaller version appears during the later phases of infection. The 24–26 kDa type was also the predominant form in viral particles. The s8ORF2 protein has previously been shown to bind RNA and interfere with interferon induction and signaling. Here we found that a fraction of the s8ORF2 protein pool in infected cells is likely to be conjugated to the interferon stimulated gene 15 (ISG15) and ubiquitin. Furthermore, several endogenous proteins pulled down by the s8ORF2 protein were identified by liquid chromatography mass spectrometry (LC-MS). PMID:26901217

  14. Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins.

    PubMed

    Olsen, Christel M; Markussen, Turhan; Thiede, Bernd; Rimstad, Espen

    2016-02-18

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus infecting salmonid fish. The virus is adapted to low temperature and has a replication optimum between 10-15 °C. In this study the subcellular localization and protein interactions for the protein encoded by the largest open reading frame of gene segment 8 (s8ORF2) were investigated. In ISAV infected cells the s8ORF2 protein was found mainly in the cytosol but a minor fraction of cells expressed the protein in the nucleus as well. Green fluorescent protein-tagged s8ORF2 did not leak out of the cell when the plasma membrane was permeabilized, suggesting interactions with intracellular structural components. The s8ORF2 protein exists both as monomer and homodimer, and co-immunoprecipitation experiments strongly suggests it binds to the ISAV fusion-, nucleo- and matrix proteins. Two versions of s8ORF2 were detected with apparent molecular weights of 24-26 and 35 kDa in lysates of infected cells. The 35 kDa type is an early viral protein while the smaller version appears during the later phases of infection. The 24-26 kDa type was also the predominant form in viral particles. The s8ORF2 protein has previously been shown to bind RNA and interfere with interferon induction and signaling. Here we found that a fraction of the s8ORF2 protein pool in infected cells is likely to be conjugated to the interferon stimulated gene 15 (ISG15) and ubiquitin. Furthermore, several endogenous proteins pulled down by the s8ORF2 protein were identified by liquid chromatography mass spectrometry (LC-MS).

  15. Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

    PubMed

    Xu, Kun; Zhang, Ting Ting; Wang, Ling; Zhang, Cun Fang; Zhang, Long; Ma, Li Xia; Xin, Ying; Ren, Chong Hua; Zhang, Zhi Qiang; Yan, Qiang; Martineau, Daniel; Zhang, Zhi Ying

    2013-02-01

    Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

  16. Avian hepatitis E virus identified in Russian chicken flocks exhibits high genetic divergence based on the ORF2 capsid gene.

    PubMed

    Sprygin, A V; Nikonova, Z B; Zinyakov, N G

    2012-10-01

    A total of 79 liver samples from clinically sick and asymptomatic chickens were tested for avian hepatitis E virus (aHEV). Samples were received from 19 farms, five of which tested positive with primers targeting the ORF2 capsid gene. The phylogenetic analysis of a 242-base-pair fragment demonstrated that the Russian aHEV isolates share between 78.2 and 96.2% over the fragment sequenced, whereas the nucleotide sequence identities between the Russian isolates and the other representatives from GeneBank varied from 76.3 to 96.2%. The homology between the studied hepatitis E viruses and swine hepatitis E virus varied between 46.9 to 48.1%. The most divergent isolate aHEV16050 showed homology of 82.6% as compared with the strains in the dendrogram. The three positive hepatitis E virus samples (aHEV16279, aHEV16050 and aHEV18196) did not cluster with the European genotype 3 as expected due to the close location of Russia to Europe, nor did they with the other two genotypes, separating to a distinct branch. The aHEV16211 grouped together with European and Chinese isolates, and the aHEV18198 with Canadian ones.

  17. Autographa californica multiple nucleopolyhedrovirus Ac92 (ORF92, P33) is required for budded virus production and multiply enveloped occlusion-derived virus formation.

    PubMed

    Wu, Wenbi; Passarelli, A Lorena

    2010-12-01

    The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C(155)XXC(158) amino acids, important for sulfhydryl oxidase activity, were mutated to A(155)XXA(158). The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype.

  18. A single amino acid substitution in the ORF1 of cymbidium ringspot virus determines the accumulation of two satellite RNAs.

    PubMed

    Rubino, Luisa; Russo, Marcello

    2012-09-01

    Tombusviruses may support the replication of satellite (sat) RNAs. In particular, two satRNAs, sat L and Cymsat RNAs, are replicated by carnation Italian ringspot (CIRV) and tomato bushy stunt (TBSV) virus, but not by cymbidium ringspot virus (CymRSV) in vitro transcripts unless they contain a poly(A) tail at the 3' end. Conversely, the replication of both satRNAs was supported by virus particles or viral RNA of the original CymRSV inoculum even in the absence of the poly(A) tail. Sequence and mutational analyses revealed that the full-length infectious CymRSV clone contains one relevant sequence variation in the ORF 1-encoded protein (p33) compared with the original inoculum, i.e. a Ser₁₉ TCC codon instead of a Phe₁₉ TTC codon, which inhibited the replication of sat L and Cymsat RNAs. It is suggested that this amino acid is contained in a domain essential for the replication of some subviral RNAs. PMID:22709553

  19. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

    PubMed Central

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

    2016-01-01

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

  20. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins.

    PubMed

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, C T; Surjit, Milan

    2016-04-26

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.

  1. Function analysis of proteins encoded by ORFs 1 to 8 of porcine circovirus-like virus P1 by microarray assay.

    PubMed

    Wen, Libin; Wang, Fengzhi; Zhang, Dan; He, Kongwang

    2015-12-01

    Porcine circovirus-like agent P1 is a newly discovered virus containing a single-strand circular genome. The genome of P1 is a DNA molecule of 648 nucleotides which contains eight open reading frames (ORFs) that probably encode potential proteins or polypeptides. Thus it is very important to clarify these proteins' function. Here we provide the methods and analysis of microarray data in detail to characterize the transcriptome profile of P1 with and without the ORF. The relevant microarray data sets have been deposited in Gene Expression Omnibus (GEO) database under accession number GSE71945. PMID:26697373

  2. Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

    PubMed

    Suzuki, Youichi; Chin, Wei-Xin; Han, Qi'En; Ichiyama, Koji; Lee, Ching Hua; Eyo, Zhi Wen; Ebina, Hirotaka; Takahashi, Hirotaka; Takahashi, Chikako; Tan, Beng Hui; Hishiki, Takayuki; Ohba, Kenji; Matsuyama, Toshifumi; Koyanagi, Yoshio; Tan, Yee-Joo; Sawasaki, Tatsuya; Chu, Justin Jang Hann; Vasudevan, Subhash G; Sano, Kouichi; Yamamoto, Naoki

    2016-01-01

    Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells. PMID:26735137

  3. Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    PubMed Central

    Ichiyama, Koji; Lee, Ching Hua; Eyo, Zhi Wen; Ebina, Hirotaka; Takahashi, Hirotaka; Takahashi, Chikako; Tan, Beng Hui; Hishiki, Takayuki; Ohba, Kenji; Matsuyama, Toshifumi; Koyanagi, Yoshio; Tan, Yee-Joo; Sawasaki, Tatsuya; Chu, Justin Jang Hann; Vasudevan, Subhash G.; Sano, Kouichi; Yamamoto, Naoki

    2016-01-01

    Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells. PMID:26735137

  4. Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

    PubMed

    Suzuki, Youichi; Chin, Wei-Xin; Han, Qi'En; Ichiyama, Koji; Lee, Ching Hua; Eyo, Zhi Wen; Ebina, Hirotaka; Takahashi, Hirotaka; Takahashi, Chikako; Tan, Beng Hui; Hishiki, Takayuki; Ohba, Kenji; Matsuyama, Toshifumi; Koyanagi, Yoshio; Tan, Yee-Joo; Sawasaki, Tatsuya; Chu, Justin Jang Hann; Vasudevan, Subhash G; Sano, Kouichi; Yamamoto, Naoki

    2016-01-01

    Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

  5. Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3.

    PubMed

    Song, D S; Yang, J S; Oh, J S; Han, J H; Park, B K

    2003-05-16

    A porcine epidemic diarrhea virus (PEDV) designated DR13 was isolated in Vero cells and serially passaged by level 100. The virus was titrated at regular intervals of the passage level. Open reading frame (ORF) 3 sequences of the virus at passage levels 20, 40, 60, 80, and 100 were aligned and compared using a computer software program. Suitability of the restriction fragment length polymorphism (RFLP) analysis for differentiating the virus from other Korean field strains was investigated. The DR13 field isolate was successively adapted in Vero cells as observed through polymerase chain reaction (PCR) and titration of the virus. RFLP analysis identified change in cleavage sites of HindIII and Xho II from passage levels 75 and 90, respectively; these RFLP patterns of ORF 3 differentiated the Vero cell-adapted virus from its parent strain, DR13, and 12 other strains of PEDV studied. The cell adapted DR13 was tested for its pathogenicity and immunogenicity in piglets and pregnant sows. The results indicated that cell adapted DR13 revealed reduced pathogenicity and induced protective immune response in pigs. Differentiation between highly Vero cell-adapted virus and wild-type virus could be the marker of adaptation to cell culture and a valuable tool for epidemiologic studies of PEDV infections. The results of this study supported that the cell attenuated virus could be applied as a marker vaccine candidate against PEDV infection.

  6. The Autographa californica multiple nucleopolyhedrovirus ORF78 is essential for budded virus production and general occlusion body formation.

    PubMed

    Tao, Xue Ying; Choi, Jae Young; Kim, Woo Jin; Lee, Joo Hyun; Liu, Qin; Kim, Song Eun; An, Saes Byeol; Lee, Seok Hee; Woo, Soo Dong; Jin, Byung Rae; Je, Yeon Ho

    2013-08-01

    ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.

  7. Hepatitis E virus ORF1 encoded macro domain protein interacts with light chain subunit of human ferritin and inhibits its secretion.

    PubMed

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-06-01

    Hepatitis E Virus (HEV) is the major causative agent of acute hepatitis in developing countries. Its genome has three open reading frames (ORFs)-called as ORF1, ORF2, and ORF3. ORF1 encodes nonstructural polyprotein having multiple domains, namely: Methyltransferase, Y domain, Protease, Macro domain, Helicase, and RNA-dependent RNA polymerase. In the present study, we show that HEV-macro domain specifically interacts with light chain subunit of human ferritin (FTL). In cultured hepatoma cells, HEV-macro domain reduces secretion of ferritin without causing any change in the expression levels of FTL. This inhibitory effect was further enhanced upon Brefeldin-A treatment. The levels of transferrin Receptor 1 or ferroportin, two important proteins in iron metabolism, remained unchanged in HEV-macro domain expressing cells. Similarly, there were no alterations in the levels of cellular labile iron pool and reactive oxygen species, indicating that HEV-macro domain does not influence cellular iron homeostasis/metabolism. As ferritin is an acute-phase protein, secreted in higher level in infected persons and HEV-macro domain has the property of reducing synthesis of inflammatory cytokines, we propose that by directly binding to FTL, macro domain prevents ferritin from entering into circulation and helps in further attenuation of the host immune response.

  8. The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1

    PubMed Central

    He, Man; Wang, Min; Huang, Ying; Peng, Wenju; Zheng, Zizheng; Xia, Ningshao; Xu, Jian; Tian, Deying

    2016-01-01

    Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity. PMID:27270888

  9. The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1.

    PubMed

    He, Man; Wang, Min; Huang, Ying; Peng, Wenju; Zheng, Zizheng; Xia, Ningshao; Xu, Jian; Tian, Deying

    2016-01-01

    Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity. PMID:27270888

  10. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus

    PubMed Central

    Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yanjin

    2016-01-01

    Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology. PMID:27548202

  11. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus.

    PubMed

    Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yanjin

    2016-01-01

    Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology. PMID:27548202

  12. Identification and function analysis of the host cell protein that interacted with Orf virus Bcl-2-like protein ORFV125.

    PubMed

    Tian, Hong; Chen, Yan; Wu, Jinyan; Lin, Tong; Liu, Xiangtao

    2016-10-01

    Orf virus (ORFV) causes contagious ecthyma, a non-systemic skin disease in sheep and goat. Bioinformatics analysis showed that ORFV125 has Bcl-2-like homologous domain and 3D structurally, it is generally known that Bcl-2 protein is known to be a key protein to control cell apoptosis. Maybe ORFV125 act as a Bcl-2-like manner to control cell apoptosis, but its exact function isn't very clear. So in this study, we use yeast two-hybrid system to identity the putative host cell protein interacting partners of ORFV125, and meanwhile using the data obtained from the Gene Ontology, Uniprot, and Kyoto Encyclopedia of Genes and Genomes databases to analysis the functions and pathways associated with them. Finally, five host proteins were shown to be interacted with ORFV125, including cytochrome b (cytb) gene, GUCY2C, BIRC5, GTF3C6 and SERBP1, we also found that BIRC5 has complex biological functions, can inhibit apoptosis, promote cell transformation and are involved in mitosis, and the interaction network of BIRC5 and ORFV125 were constructed. These findings provide a foundation to better understand the biology of the interactions between ORFV125 and the host proteins with which it directly interacts with and resultant downstream events. PMID:27663376

  13. Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity.

    PubMed

    Couñago, Rafael M; Knapp, Karen M; Nakatani, Yoshio; Fleming, Stephen B; Corbett, Michael; Wise, Lyn M; Mercer, Andrew A; Krause, Kurt L

    2015-07-01

    The chemokine binding protein (CKBP) from orf virus (ORFV) binds with high affinity to chemokines from three classes, C, CC, and CXC, making it unique among poxvirus CKBPs described to date. We present its crystal structure alone and in complex with three CC chemokines, CCL2, CCL3, and CCL7. ORFV CKBP possesses a β-sandwich fold that is electrostatically and sterically complementary to its binding partners. Chemokines bind primarily through interactions involving the N-terminal loop and a hydrophobic recess on the ORFV CKBP β-sheet II surface, and largely polar interactions between the chemokine 20s loop and a negatively charged surface groove located at one end of the CKBP β-sheet II surface. ORFV CKBP interacts with leukocyte receptor and glycosaminoglycan binding sites found on the surface of bound chemokines. SEC-MALLS and chromatographic evidence is presented supporting that ORFV CKBP is a dimer in solution over a broad range of protein concentrations. PMID:26095031

  14. African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase.

    PubMed

    Coelho, João; Martins, Carlos; Ferreira, Fernando; Leitão, Alexandre

    2015-01-01

    Topoisomerases modulate the topological state of DNA during processes, such as replication and transcription, that cause overwinding and/or underwinding of the DNA. African swine fever virus (ASFV) is a nucleo-cytoplasmic double-stranded DNA virus shown to contain an OFR (P1192R) with homology to type II topoisomerases. Here we observed that pP1192R is highly conserved among ASFV isolates but dissimilar from other viral, prokaryotic or eukaryotic type II topoisomerases. In both ASFV/Ba71V-infected Vero cells and ASFV/L60-infected pig macrophages we detected pP1192R at intermediate and late phases of infection, cytoplasmically localized and accumulating in the viral factories. Finally, we used a Saccharomyces cerevisiae temperature-sensitive strain in order to demonstrate, through complementation and in vitro decatenation assays, the functionality of P1192R, which we further confirmed by mutating its predicted catalytic residue. Overall, this work strengthens the idea that P1192R constitutes a target for studying, and possibly controlling, ASFV transcription and replication.

  15. Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

    SciTech Connect

    Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

    2010-09-30

    Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

  16. Expression of the Murine Norovirus (MNV) ORF1 Polyprotein Is Sufficient to Induce Apoptosis in a Virus-Free Cell Model

    PubMed Central

    Skilton, Rachel J.; Prince, Cynthia A.; Ward, Vernon K.; Lambden, Paul R.; Clarke, Ian N.

    2014-01-01

    Investigations into human norovirus infection, replication and pathogenesis, as well as the development of potential antiviral agents, have been restricted by the lack of a cell culture system for human norovirus. To date, the optimal cell culture surrogate virus model for studying human norovirus biology is the murine norovirus (MNV). In this report we generate a tetracycline-regulated, inducible eukaryotic cell system expressing the entire MNV ORF1 polyprotein. Once induced, the MNV ORF1 polyprotein was faithfully processed to the six mature non-structural proteins that predominately located to a discrete perinuclear region, as has been observed in active MNV infection. Furthermore, we found that expression of the ORF1 polyprotein alone was sufficient to induce apoptosis, characterised by caspase-9 activation and survivin down-regulation. This cell line provides a valuable new tool for studying MNV ORF1 non-structural protein function, screening for potential antiviral agents and acts as a proof-of-principle for such systems to be developed for human noroviruses. PMID:24599381

  17. Expression of the murine norovirus (MNV) ORF1 polyprotein is sufficient to induce apoptosis in a virus-free cell model.

    PubMed

    Herod, Morgan R; Salim, Omar; Skilton, Rachel J; Prince, Cynthia A; Ward, Vernon K; Lambden, Paul R; Clarke, Ian N

    2014-01-01

    Investigations into human norovirus infection, replication and pathogenesis, as well as the development of potential antiviral agents, have been restricted by the lack of a cell culture system for human norovirus. To date, the optimal cell culture surrogate virus model for studying human norovirus biology is the murine norovirus (MNV). In this report we generate a tetracycline-regulated, inducible eukaryotic cell system expressing the entire MNV ORF1 polyprotein. Once induced, the MNV ORF1 polyprotein was faithfully processed to the six mature non-structural proteins that predominately located to a discrete perinuclear region, as has been observed in active MNV infection. Furthermore, we found that expression of the ORF1 polyprotein alone was sufficient to induce apoptosis, characterised by caspase-9 activation and survivin down-regulation. This cell line provides a valuable new tool for studying MNV ORF1 non-structural protein function, screening for potential antiviral agents and acts as a proof-of-principle for such systems to be developed for human noroviruses. PMID:24599381

  18. Autographa californica multiple nucleopolyhedrovirus orf114 is not essential for virus replication in vitro, but its knockout reduces per os infectivity in vivo.

    PubMed

    Wei, Wenqiang; Zhou, Yin; Lei, Chengfeng; Sun, Xiulian

    2012-10-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf114 (ac114) is one of the highly conserved unique genes in the lepidopteran group I nucleopolyhedrovirus. So far, the biological function of ac114 is unknown. To study the function of ac114 in the virus life cycle, an ac114 knockout baculovirus shuttle vector (bacmid) was generated. Fluorescence and light microscopy showed that the ac114 knockout mutant was able to produce infectious budded viruses (BVs) and occlusion bodies (OBs). Titration assays demonstrated that the ac114 knockout virus had similar growth kinetics to the control virus during the infection phase. Electron microscopy indicated that ac114 did not affect the morphogenesis of BVs and occlusion-derived viruses (ODVs); however, the numbers of ODVs per OB of the ac114 knockout virus were significantly lower than those of the control virus. RT-PCR demonstrated that ac114 was a late stage expression gene and that its transcription initiated at an A residue, 16 nucleotides upstream of the ATG start codon. Intracellular localization analysis revealed that the Ac114-GFP fusion protein localized predominantly as punctate patches in the cytoplasm of infected Sf9 cells. Bioassays showed that the ac114 knockout did not change the killing speed of AcMNPV in Spodoptera exigua larvae, but reduced its viral infectivity significantly. Taken together, these data indicate that ac114 is an auxiliary gene that facilitates embedding of ODVs into OBs, thus affecting the per os infectivity of the virus.

  19. Genome sequence heterogeneity of Lake Sinai Virus found in honey bees and Orf1/RdRP-based polymorphisms in a single host.

    PubMed

    Ravoet, Jorgen; De Smet, Lina; Wenseleers, Tom; de Graaf, Dirk C

    2015-04-01

    Honey bees (Apis mellifera) are susceptible to a wide range of pathogens, including a broad set of viruses. Recently, next-generation sequencing has expanded the list of viruses with, for instance, two strains of Lake Sinai Virus. Soon after its discovery in the USA, LSV was also discovered in other countries and in other hosts. In the present study, we assemble four almost complete LSV genomes, and show that there is remarkable sequence heterogeneity based on the Orf1, RNA-dependent RNA polymerase and capsid protein sequences in comparison to the previously identified LSV 1 and 2 strains. Phylogenetic analyses of LSV sequences obtained from single honey bee specimens further revealed that up to three distinctive clades could be present in a single bee. Such superinfections have not previously been identified for other honey bee viruses. In a search for the putative routes of LSV transmission, we were able to demonstrate the presence of LSV in pollen pellets and in Varroa destructor mites. However, negative-strand analyses demonstrated that the virus only actively replicates in honey bees and mason bees (Osmia cornuta) and not in Varroa mites.

  20. An African swine fever virus ORF with similarity to C-type lectins is non-essential for growth in swine macrophages in vitro and for virus virulence in domestic swine.

    PubMed

    Neilan, J G; Borca, M V; Lu, Z; Kutish, G F; Kleiboeker, S B; Carrillo, C; Zsak, L; Rock, D L

    1999-10-01

    An African swine fever virus (ASFV) ORF, 8CR, with similarity to the C-type lectin family of adhesion proteins has been described in the pathogenic isolate Malawi Lil-20/1. The similarity of 8CR to cellular and poxvirus genes associated with cell adhesion, cell recognition and virus infectivity suggested that 8CR may be of significance to ASFV-host cell interactions. Sequence analysis of the 8CR ORF from additional pathogenic ASFV isolates demonstrated conservation among isolates from both pig and tick sources. Northern blot analysis demonstrated 8CR mRNA transcription late in the virus replication cycle. A Malawi Lil-20/1 8CR deletion mutant (delta8CR) was constructed to analyse 8CR function further. The growth characteristics in vitro of delta8CR in porcine macrophage cell cultures were identical to those observed for parental virus. In domestic swine, delta8CR exhibited an unaltered parental Malawi Lil-20/1 disease and virulence phenotype. Thus, although well conserved among pathogenic ASFV field isolates, 8CR is non-essential for growth in porcine macrophages in vitro and for virus virulence in domestic swine.

  1. Recombinant lactobacillus expressing G protein of spring viremia of carp virus (SVCV) combined with ORF81 protein of koi herpesvirus (KHV): A promising way to induce protective immunity against SVCV and KHV infection in cyprinid fish via oral vaccination.

    PubMed

    Cui, Li-Chun; Guan, Xue-Ting; Liu, Zhong-Mei; Tian, Chang-Yong; Xu, Yi-Gang

    2015-06-17

    Spring viremia of carp virus (SVCV) and koi herpesvirus (KHV) are highly contagious and pathogenic to cyprinid fish, causing enormous economic losses in aquaculture. Although DNA vaccines reported in recent years could induce protective immune responses in carps against these viruses via injection, there are a number of consequences and uncertainties related to DNA vaccination. Therefore, more effective and practical method to induce protective immunity such as oral administration would be highly desirable. In this study, we investigated the utilities of a genetically engineered Lactobacillus plantarum (L. plantarum) coexpressing glycoprotein (G) of SVCV and ORF81 protein of KHV as oral vaccine to induce protective immunity in carps via oral vaccination. The surface-displayed recombinant plasmid pYG-G-ORF81 was electroporated into L. plantarum, giving rise to LP/pYG-G-ORF81, where expression and localization of G-ORF81 fusion protein from the LP/pYG-G-ORF81 was identified by SDS-PAGE, Western blotting and immunofluorescence assay. Bait feed particles containing the LP/pYG-G-ORF81 were used as vaccine to immunize carps via gastrointestinal route. Compared to control groups, the carps orally immunized with the LP/pYG-G-ORF81 were induced significant levels of immunoglobulin M (IgM), and its immunogenicity was confirmed by viral loads reduction detected by PCR assay after virus challenge followed by an effective protection rate 71% in vaccinated carps and 53% in vaccinated koi until at days 65 post challenge, respectively. Our study here demonstrates, for the first time, the ability of recombinant L. plantarum as oral vaccine against SVCV and KHV infection in carps, suggesting a practical multivalent strategy for the control of spring viremia of carp and koi herpesvirus disease. PMID:25981489

  2. Maternal transfer of antibodies to the offspring after mice immunization with insect larvae-derived recombinant hepatitis E virus ORF-2 proteins.

    PubMed

    Jiménez de Oya, Nereida; Alonso-Padilla, Julio; Blázquez, Ana-Belén; Escribano-Romero, Estela; Escribano, José M; Saiz, Juan-Carlos

    2011-06-01

    Hepatitis E virus (HEV) is a major cause of acute hepatitis in humans, causing outbreaks and epidemics in regions with sub-optimal sanitary conditions, in many of which it is endemic. Nowadays there is no specific therapy or licensed vaccines against HEV infection. In this study, we have analyzed in mice the immunogenicity of HEV open-reading frame 2 (ORF-2) protein, and a truncated form of it lacking the first 111 amino acids, efficiently expressed in an improved baculovirus-based technology using insects as living biofactories. Both recombinant proteins elicited high and long-lasting specific anti HEV antibodies. Passive transfer of immunity from immunized mothers to their offspring was demonstrated to occur both by transplacental and lactation routes. These results indicate that these insect-derived immunogens constitute low-cost potential vaccine candidate to be further evaluated.

  3. Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles.

    PubMed

    Lankester, Felix; Lugelo, Ahmed; Mnyambwa, Nicholas; Ndabigaye, Ahab; Keyyu, Julius; Kazwala, Rudovick; Grant, Dawn M; Relf, Valerie; Haig, David M; Cleaveland, Sarah; Russell, George C

    2015-01-01

    Alcelaphine herpesvirus-1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF. PMID:25969987

  4. Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles

    PubMed Central

    Lankester, Felix; Lugelo, Ahmed; Mnyambwa, Nicholas; Ndabigaye, Ahab; Keyyu, Julius; Kazwala, Rudovick; Grant, Dawn M.; Relf, Valerie; Haig, David M.; Cleaveland, Sarah; Russell, George C.

    2015-01-01

    Alcelaphine herpesvirus–1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF. PMID:25969987

  5. Phylogenetics based on partial ORF2 of triatoma virus in triatomines collected over a decade from domiciliary habitats.

    PubMed

    Susevich, María Laura; Marti, Gerardo Aníbal; Balsalobre, Agustín; Echeverría, María Gabriela

    2015-01-01

    The only virus sequenced and studied in triatomines is the Triatoma virus, from the Dicistroviridae family, which causes delayed development, reduced oviposition, and premature death of infected insects. With the goal of expanding the sequences already obtained in previous years and verifying if any changes occurred in their genomic sequences, 68 samples of triatomines from several provinces of Argentina were analyzed. Sixteen positive samples were obtained by Reverse Transcription (RT)-polymerase chain reaction using the VP3-VP1 subregion of open reading frame-2 as a diagnostic method; after sequencing, 11 samples were obtained from Triatoma infestans. These new sequences showed no significant differences in the analyzed regions, which were not grouped by species or habitat or geographical distribution. There were no differences when compared with the sequences found during 2002-2012, all obtained from the wild. We conclude that despite being an RNA virus, the different sequences show high homology. PMID:25797795

  6. Phylogenetics Based on Partial ORF2 of Triatoma Virus in Triatomines Collected Over a Decade From Domiciliary Habitats

    PubMed Central

    Susevich, María Laura; Marti, Gerardo Aníbal; Balsalobre, Agustín; Echeverría, María Gabriela

    2015-01-01

    The only virus sequenced and studied in triatomines is the Triatoma virus, from the Dicistroviridae family, which causes delayed development, reduced oviposition, and premature death of infected insects. With the goal of expanding the sequences already obtained in previous years and verifying if any changes occurred in their genomic sequences, 68 samples of triatomines from several provinces of Argentina were analyzed. Sixteen positive samples were obtained by Reverse Transcription (RT)-polymerase chain reaction using the VP3-VP1 subregion of open reading frame-2 as a diagnostic method; after sequencing, 11 samples were obtained from Triatoma infestans. These new sequences showed no significant differences in the analyzed regions, which were not grouped by species or habitat or geographical distribution. There were no differences when compared with the sequences found during 2002–2012, all obtained from the wild. We conclude that despite being an RNA virus, the different sequences show high homology. PMID:25797795

  7. Effects of Early or Overexpression of the Autographa californica Multiple Nucleopolyhedrovirus orf94 (ODV-e25) on Virus Replication.

    PubMed

    Luo, Xiao-Chun; Wang, Shan-Shan; Zhang, Jie; Qian, Duo-Duo; Wang, Si-Min; Li, Lu-Lin

    2013-01-01

    odv-e25(e25) is one of the core genes of baculoviruses. To investigate how it functions in the replication cycle of a baculovirus, a number of Autographa californica multiple nucleopolyhedrovirus recombinants with e25 under control of the promoter of immediate early gene ie1, or the promoter of the very late hyperexpressed gene p10, were constructed using a bacmid system, and the effects of early expression or overexpression of e25 on replication of the virus were evaluated. Microscopy and titration assays demonstrated that bacmids with e25 under control of ie1 promoter were unable to produce budded viruses; and that the recombinant viruses with e25 under control of p10 promoter generated budded virus normally, but formation of occlusion bodies were dramatically reduced and delayed in the infected cells. Electron microscopy showed that there were no mature virions or intact nucleocapsids present in the cells transfected with a recombinant bacmid with e25 under control of ie1 promoter. Quantitative real-time PCR analysis demonstrated that alteration of the e25 promoter did not affect viral DNA synthesis. The reporter gene expression from the promoter of the major capsid protein gene vp39 was reduced 63% by early expression of e25. Confocal microscopy revealed that E25 was predominantly localized in nuclei by 24 hours post infection with wild-type virus, but it remained in the cytoplasm in the cells transfected with a recombinant bacmid with e25 under control of the ie1 promoter, suggesting that the transport of E25 into nuclei was regulated in a specific and strict time dependent manner.

  8. 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

    PubMed Central

    Wirblich, C; Sibilia, M; Boniotti, M B; Rossi, C; Thiel, H J; Meyers, G

    1995-01-01

    Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size. PMID:7474137

  9. Molecular Characterization of the ORF3 and S1 Genes of Porcine Epidemic Diarrhea Virus Non S-INDEL Strains in Seven Regions of China, 2015

    PubMed Central

    Wang, Enyu; Guo, Donghua; Li, Chunqiu; Wei, Shan; Wang, Zhihui; Liu, Qiujin; Zhang, Bei; Kong, Fanzhi; Feng, Li; Sun, Dongbo

    2016-01-01

    In an effort to trace the evolution of porcine epidemic diarrhea virus (PEDV), S1 and ORF3 genes of viruses identified in 41 pig farms from seven regions (North, Northeast, Northwest, Central, East, South West, and South, respectively) of China in 2015 were sequenced and analyzed. Sequence analysis revealed that the 41 ORF3 genes and 29 S1 genes identified in our study exhibited nucleotide homologies of 98.2%–100% and 96.6%–100%, respectively; these two genes exhibited low nucleotide sequence similarities with classical CV777 strain and early Chinese strain LZC. Phylogenetic analysis indicated that the identified PEDV strains belonged to global non S-INDEL strains, and exhibited genetic diversity; S1 gene of the HLJ2015/DP1-1 strain harbored an unique deletion of 12 nucleotides (A1130CAACTCCACTG1141); while the Chinese PEDV S-INDEL reference strains included two types of the “CV777” S-INDEL as well as the “US” S-INDEL, and all co-circulated with Chinese non S-INDEL strains. Of 29 identified S1 genes, the SS2 epitope (Y748SNIGVCK755) was highly conserved, while the SS6 epitope (L764QDGQVKI771) and pAPN receptor-binding region (aa 490–615) exhibited amino substitutions. Nine possible recombination events were identified between the 29 identifed S1 genes and the 3 S1 reference genes from early Chinese PEDV strains. The complete S genes of selected Chinese PEDV field strains (2011–2015) showed 5.18%–6.07% nucleotide divergence, which is far higher than the divergence observed in early Chinese PEDV strains (3.1%) (P<0.05). Our data provide evidence that PEDV non S-INDEL strains with genetic diversities and potential recombination circulate in seven regions of China in 2015; Chinese PEDV S-INDEL strains exhibit genetic diversity and co-circulate with non S-INDEL strains. PMID:27494026

  10. Silencing of ORFs C2 and C4 of Tomato Yellow Leaf Curl Virus Engenders Resistant or Tolerant Plants.

    PubMed

    Peretz, Yuval; Eybishtz, Assaf; Sela, Ilan

    2011-01-01

    The IL-60 system is a transient universal vector system for expression and silencing in plants [1]. This vector has been derived from Tomato yellow leaf curl virus (TYLCV). The viral intergenic region (IR) is a non-coding short (314 b) sequence separating the viral sense-oriented genes from the complementary-oriented genes. IR carries the viral origin of replication as well as a promoter at each end. Placing a gene segment between two IRs at opposite orientations followed by trans-activation of the construct by the plasmid IL-60-BS, caused silencing of the pertinent gene as indicated by the silencing of the endogenous gene PDS.. The viral genes C2 and C4 are implicated as having a role in viral-directed silencing suppression. The silencing of C2 and C4 intervened with the virus ability to counter-react to viral silencing by the host plant, thus engendering resistance or tolerance. PMID:22253651

  11. Transgenic Expression of Walleye Dermal Sarcoma Virus rv-cyclin (orfA) in Zebrafish does not Result in Tissue Proliferation

    PubMed Central

    Paul, Thomas A.; Rovnak, Joel; Quackenbush, Sandra L.; Whitlock, Kathleen; Zhan, Huiqing; Gong, Zhiyuan; Spitsbergen, Jan; Bowser, Paul R.

    2012-01-01

    Walleye dermal sarcoma (WDS) is a benign tumor of walleye fish that develops and completely regresses seasonally. The retrovirus associated with this disease, walleye dermal sarcoma virus, encodes three accessory genes, two of which, rv-cyclin (orfA) and orfb, are thought to play a role in tumor development. In this study, we attempted to recapitulate WDS development by expressing rv-cyclin in chimeric and stable transgenic zebrafish. Six stable transgenic lines expressing rv-cyclin from the constitutive CMVtk promoter were generated. Immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction demonstrate that rv-cyclin is widely expressed in different tissues in these fish. These lines were viable and histologically normal for up to 2 years. No increase in tumors or tissue proliferation was observed following N-ethyl N-nitrosourea exposure or following tail wounding and subsequent tissue regeneration compared to controls. These data indicate that rvcyclin is not independently sufficient for tumor induction in zebrafish. PMID:20349325

  12. The Adenovirus Type 5 E1B-55K Oncoprotein Actively Shuttles in Virus-Infected Cells, Whereas Transport of E4orf6 Is Mediated by a CRM1-Independent Mechanism

    PubMed Central

    Dosch, Tanja; Horn, Florian; Schneider, Grit; Krätzer, Friedrich; Dobner, Thomas; Hauber, Joachim; Stauber, Roland H.

    2001-01-01

    The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the “driving force” for adenoviral late mRNA export. PMID:11356976

  13. Visceral leishmaniosis and parapoxvirus infection in a Mediterranean monk seal (Monachus monachus).

    PubMed

    Toplu, N; Aydoğan, A; Oguzoglu, T C

    2007-05-01

    A Mediterranean monk seal was shown by immunohistochemical and polymerase chain reaction techniques to be dually infected with a Leishmania sp. and parapoxvirus. The pathological findings included a deep ulcer on the side of the head, ulcers on the gingival and inner aspect of the lower lip, enlarged lymph nodes and tonsils, and respiratory lesions (pulmonary consolidation, oedema, haemorrhages and emphysema; tracheal and bronchial congestion, exudates and haemorrhage). Amastigotes were demonstrated in macrophages in the lymph nodes and spleen, and intracytoplasmic inclusion bodies were observed in the tracheal and oral mucosa. PMID:17459405

  14. Variation in Fetal Outcome, Viral Load and ORF5 Sequence Mutations in a Large Scale Study of Phenotypic Responses to Late Gestation Exposure to Type 2 Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Ladinig, Andrea; Wilkinson, Jamie; Ashley, Carolyn; Detmer, Susan E.; Lunney, Joan K.; Plastow, Graham; Harding, John C. S.

    2014-01-01

    In spite of extensive research, the mechanisms of reproductive disease associated with Porcine Reproductive and Respiratory Syndrome virus (PRRSv) are still poorly understood. The objectives of this large scale study were to evaluate associations between viral load and fetal preservation, determine the impact of type 2 PRRSv on fetal weights, and investigate changes in ORF5 PRRSv genome in dams and fetuses during a 21-day period following challenge. At gestation day 85 (±1), 114 gilts were experimentally infected with type 2 PRRSv, while 19 gilts served as reference controls. At necropsy, fetuses were categorized according to their preservation status and tissue samples were collected. PRRSv RNA concentrations were measured in gilt serum collected on days 0, 2, 6, and 21 post-infection, as well as in gilt and fetal tissues collected at termination. Fetal mortality was 41±22.8% in PRRS infected litters. Dead fetuses appeared to cluster in some litters but appeared solitary or random in others. Nine percent of surviving piglets were meconium-stained. PRRSv RNA concentration in fetal thymus, fetal serum and endometrium differed significantly across preservation category and was greatest in tissues of meconium-stained fetuses. This, together with the virtual absence of meconium staining in non-infected litters indicates it is an early pathological condition of reproductive PRRS. Viral load in fetal thymus and in fetal serum was positively associated with viral load in endometrium, suggesting the virus exploits dynamic linkages between individual maternal-fetal compartments. Point mutations in ORF5 sequences from gilts and fetuses were randomly located in 20 positions in ORF5, but neither nucleotide nor amino acid substitutions were associated with fetal preservation. PRRSv infection decreased the weights of viable fetuses by approximately 17%. The considerable variation in gilt and fetal outcomes provides tremendous opportunity for more detailed investigations of

  15. Efficacy of a Parapoxvirus ovis-based immunomodulator against equine herpesvirus type 1 and Streptococcus equi equi infections in horses.

    PubMed

    Ons, Ellen; Van Brussel, Leen; Lane, Stephen; King, Vickie; Cullinane, Ann; Kenna, Rachel; Lyons, Pamela; Hammond, Toni-Ann; Salt, Jeremy; Raue, Rudiger

    2014-10-10

    The efficacy of Zylexis®, an immunomodulator in horses based on inactivated Parapoxvirus ovis (iPPVO), was assessed using an equine herpesvirus type 1 (EHV-1) challenge model in the presence of a natural infection with Streptococcus equi equi (S. equi). Eleven horses were treated with iPPVO and twelve were kept as controls. Six horses were challenged with EHV-1 and commingled with the horses on study. Animals were dosed on Days -2, 0 (just before commingling) and Day 7. On Day 11 significantly less nasal discharge, enlarged lymph nodes, EHV-1 shedding and lower rectal temperatures were observed in the iPPVO-treated group. In addition, iPPVO-treated horses showed significantly fewer enlarged lymph nodes on Days 17 and 19, significantly less lower jaw swelling on Day 3 and significantly lower rectal temperatures on Days 12 and 13. Dyspnoea, depression and anorexia were only recorded for the control group. Following challenge seven out of 11 horses in the iPPVO treated group shed EHV-1 but on Days 11, 12, 13, 14, 15 and 16 quantitative virus detection in this group was significantly lower as compared to the controls. All animals shed S. equi but the percentage of animals with positive bacterial detection was lower in the iPPVO group than in the control group from Day 14 through Day 28. This difference was significant on Day 24. No injection site reactions or adverse events were observed. In conclusion, Zylexis administration is safe and reduced clinical signs and shedding related to both EHV-1 and S. equi infections.

  16. Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein.

    PubMed

    Liu, D X; Brown, T D

    1995-06-01

    Coronavirus gene expression involves proteolytic processing of the mRNA 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF 1b-encoded 100-kDa protein is proteolytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus 3C proteinase group (3C-like proteinase). In this report, the 3C-like proteinase has been further analysed by internal deletion of a 2.3-kb fragment between the 3C-like proteinase-encoding region and ORF 1b and by substitution mutations of its catalytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF 1a sequences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to the 100-kDa protein species. Site-directed mutagenesis studies have confirmed that the predicted nucleophilic cysteine residue (Cys2922) and a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 (His2820) are essential for the catalytic activity of the proteinase, and that the QS(G) dipeptide bonds are its target cleavage sites. Substitution mutations of the third component of the putative catalytic triad, the glutamic acid 2843 (Glu2843) residue, however, do not affect the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the 3C-like proteinase is capable of trans-cleavage of the 1a/1b polyprotein. These studies have confirmed the involvement of the 3C-like proteinase domain in processing of the 1a/1b polyprotein, the predicted catalytic centre of the proteinase, and its cleavage sites. PMID:7778277

  17. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    SciTech Connect

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  18. Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly

    PubMed Central

    Wang, Fan; Liu, Yang; Zhu, Yi; Ngoc Tran, Bich; Wu, Jinlu; Leong Hew, Choy

    2015-01-01

    Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, and the low yield of infectious particles. Comparative proteomics further identified the structural proteins affected by ORF75R knockdown. Two-dimensional gel electrophoresis combined with proteomics demonstrated that ORF75R was phosphorylated at multiple sites in SGIV-infected cell lysate and virions, but the vast majority of ORF75R in virions was the dephosphorylated isoform. A kinase assay showed that ORF75R could be phosphorylated in vitro by the SGIV structural protein ORF39L. Addition of ATP and Mg2+ into purified virions prompted extensive phosphorylation of structural proteins and release of ORF75R from virions. These data suggest that ORF75R is a novel scaffold protein important for viral assembly and DNA encapsidation, but its phosphorylation facilitates virion disassembly. Compared to proteins from other viruses, we found that ORF75R shares common features with herpes simplex virus VP22. PMID:26286371

  19. The Ep152R ORF of African swine fever virus strain Georgia encodes for an essential gene that interacts with host protein BAG6.

    PubMed

    Borca, Manuel V; O'Donnell, Vivian; Holinka, Lauren G; Rai, Devendra K; Sanford, Brenton; Alfano, Marialexia; Carlson, Jolene; Azzinaro, Paul A; Alonso, Covadonga; Gladue, Douglas P

    2016-09-01

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few of these genes have been studied in some detail. Here we report the characterization of open reading frame Ep152R that has a predicted complement control module/SCR domain. This domain is found in Vaccinia virus proteins that are involved in blocking the immune response during viral infection. A recombinant ASFV harboring a HA tagged version of the Ep152R protein was developed (ASFV-G-Ep152R-HA) and used to demonstrate that Ep152R is an early virus protein. Attempts to construct recombinant viruses having a deleted Ep152R gene were consistently unsuccessful indicating that Ep152R is an essential gene. Interestingly, analysis of host-protein interactions for Ep152R using a yeast two-hybrid screen, identified BAG6, a protein previously identified as being required for ASFV replication. Furthermore, fluorescent microscopy analysis confirms that Ep152R-BAG6 interaction actually occurs in cells infected with ASFV. PMID:27497620

  20. The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential gene that interacts with host protein BAG6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few have been studied in some detail. Here we rep...

  1. Inactivated Parapoxvirus ovis as inducer of immunity in silver catfish (Rhamdia quelen).

    PubMed

    Pavan, Tatiana R; Nied, Cristian O; Noro, Mirela; Anziliero, Deniz; Frandoloso, Rafael; Kreutz, Luiz Carlos

    2016-09-01

    Molecules with immune modulating activity are ubiquitously distributed in nature and their impact on aquaculture has been exploited in order to increase fish resistance to pathogens. Here, we investigated the effect of inactivated Parapoxvirus ovis (iPPVO) on blood cells and innate and acquired immune response of silver catfish (Rhamdia quelen). iPPVO inoculation had no effect on respiratory burst activity; however, following iPPVO inoculation, we observed a significant decrease on circulating monocytes concomitantly with an increased number of heterophilic granulocytes and thrombocytes, which are the main cells involved in innate immunity and provide connection with acquired immunity. Fish inoculated with a combination of bovine serum albumin (BSA) + iPPVO had significantly higher levels of antibodies to BSA compared to fish inoculated with BSA alone, but lower than fish inoculated with BSA + Freund's incomplete adjuvant (FIA). These findings points to the potential usefulness of iPPVO as immunomodulator in fish and instigate further research to identify its component that interact with immune cells and that could be exploited as adjuvants in fish.

  2. Efficacy of amitraz plus inactivated parapoxvirus ovis in the treatment of canine generalised demodicosis.

    PubMed

    Pekmezci, D; Pekmezci, G Z; Guzel, M; Cenesiz, S; Gurler, A T; Gokalp, G

    2014-05-31

    Canine generalised demodicosis (CGD) is a challenging disease to treat effectively. Inactivated parapoxvirus ovis (iPPVO) could help to accelerate treatment with acaricidial therapy by altering the immune response. This study was designed to investigate the effects of treating CGD with amitraz plus iPPVO in terms of clinical outcomes and blood parameters. The study involved 16 dogs ranging in age from eight months to six years and weighing between 10 and 40 kg. Eight dogs were treated with amitraz and eight with amitraz plus iPPVO. Biochemical analysis of whole blood and serum, including serum C reactive protein (CRP) and serum amyloid A (SAA), was performed. Skin scrapings were conducted on days 0, 10, 40, 80 and 120 of treatment, and mite numbers were recorded. Clinical remission was determined according to mite numbers and clinical scores. The difference in mean whole remission days between the amitraz group (104.3 days) and the amitraz+iPPVO group (84.5 days) was statistically significant (P<0.05). Mean clinical scores were also significantly better in the amitraz+iPPVO (5.60) group when compared with the amitraz group (7.65). No adverse reactions were observed in either group. In view of these findings, the use of iPPVO in conjunction with amitraz can be recommended for treating CGD.

  3. CyHV-2 ORF104 activates the p38 MAPK pathway.

    PubMed

    Du, Mi; Chen, Mingliang; Shen, Haifeng; Wang, Wei; Li, Zengpeng; Wang, Weiyi; Huang, Jianhui; Chen, Jianming

    2015-10-01

    Cyprinid herpesvirus 2 (CyHV-2) is the pathogen responsible for herpesviral hematopoietic necrosis disease, which causes huge losses on aquaculture. So far the studies of CyHV-2 mainly focus on the identification and detection of this virus, but little is known about the role of specific CyHV-2 genes in the infection process. Based on the genomic information, CyHV-2 ORF104 encodes a kinase-like protein, which is highly conserved among the three CyHVs. Our study was initiated to investigate the role of kinase-like protein ORF104 during virus infection. Subcellular localization study showed that ORF104 was mainly expressed in the nucleus in both human HEK293T and fish EPC cells. However, deletion of the putative nuclear localization signal of ORF104 (ORF104M) resulted in the cytoplasmic distribution in HEK293T. We then examined whether MAPKs were involved in the ORF104-mediated signaling pathway by overexpressing ORF104 and ORF104M in HEK293T. Overexpression of ORF104 and ORF104M resulted in the up-regulation of p38 phosphorylation, but not JNK or ERK, indicating that ORF104 specifically activates p38 signaling pathway. In vivo study showed that CyHV-2 infection enhanced p38 phosphorylation in gibel carp (Carassius auratus gibelio). Interestingly, p38 inhibitor SB203580 strongly reduced fish death caused by CyHV-2 infection. Therefore, our study for the first time reveals the function of ORF104 during CyHV-2 infection, indicating that ORF104 is a potential vaccine candidate for CyHV-2.

  4. Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin.

    PubMed

    Lv, Qizhuang; Guo, Kangkang; Wang, Tao; Zhang, Chengcheng; Zhang, Yanming

    2015-09-01

    Porcine circovirus type 2 (PCV2) is the primary infectious agent of PCV-associated disease (PCVAD) in swine. ORF4 protein is a newly identified viral protein of PCV2 and is involved in virus-induced apoptosis. However, the molecular mechanisms of ORF4 protein regulation of apoptosis remain unclear, especially given there is no information regarding any cellular partners of the ORF4 protein. Here, we have utilized the yeast two-hybrid assay and identified four host proteins (FHC, SNRPN, COX8A and Lamin C) interacting with the ORF4 protein. Specially, FHC was chosen for further characterization due to its important role in apoptosis. GST pull-down, subcellular co-location and co-immunoprecipitation assays confirmed that the PCV2 ORF4 protein indeed interacted with the heavy-chain ferritin, which is an interesting clue that will allow us to determine the role of the ORF4 protein in apoptosis. PMID:26333394

  5. Genomic sequence of mandarin fish rhabdovirus with an unusual small non-transcriptional ORF.

    PubMed

    Tao, Jian-Jun; Zhou, Guang-Zhou; Gui, Jian-Fang; Zhang, Qi-Ya

    2008-03-01

    The complete genome of mandarin fish Siniperca chuatsi rhabdovirus (SCRV) was cloned and sequenced. It comprises 11,545 nucleotides and contains five genes encoding the nucleoprotein N, the phosphoprotein P, the matrix protein M, the glycoprotein G, and the RNA-dependent RNA polymerase protein L. At the 3' and 5' termini of SCRV genome, leader and trailer sequences show inverse complementarity. The N, P, M and G proteins share the highest sequence identities (ranging from 14.8 to 41.5%) with the respective proteins of rhabdovirus 903/87, the L protein has the highest identity with those of vesiculoviruses, especially with Chandipura virus (44.7%). Phylogenetic analysis of L proteins showed that SCRV clustered with spring vireamia of carp virus (SVCV) and was most closely related to viruses in the genus Vesiculovirus. In addition, an overlapping open reading frame (ORF) predicted to encode a protein similar to vesicular stomatitis virus C protein is present within the P gene of SCRV. Furthermore, an unoverlapping small ORF downstream of M ORF within M gene is predicted (tentatively called orf4). Therefore, the genomic organization of SCRV can be proposed as 3' leader-N-P/C-M-(orf4)-G-L-trailer 5'. Orf4 transcription or translation products could not be detected by northern or Western blot, respectively, though one similar mRNA band to M mRNA was found. This is the first report on one small unoverlapping ORF in M gene of a fish rhabdovirus.

  6. Novel histone H3 binding protein ORF158L from the Singapore grouper iridovirus.

    PubMed

    Tran, Bich Ngoc; Chen, Liming; Liu, Yang; Wu, Jinlu; Velázquez-Campoy, Adrián; Sivaraman, J; Hew, Choy Leong

    2011-09-01

    Singapore grouper iridovirus (SGIV), a major pathogen of concern for grouper aquaculture, has a double-stranded DNA (dsDNA) genome with 162 predicted open reading frames, for which a total of 62 SGIV proteins have been identified. One of these, ORF158L, bears no sequence homology to any other known protein. Knockdown of orf158L using antisense morpholino oligonucleotides resulted in a significant decrease in virus yield in grouper embryonic cells. ORF158L was observed in nuclei and virus assembly centers of virus-infected cells. This observation led us to study the structure and function of ORF158L. The crystal structure determined at 2.2-Å resolution reveals that ORF158L partially exhibits a structural resemblance to the histone binding region of antisilencing factor 1 (Asf1), a histone H3/H4 chaperon, despite the fact that there is no significant sequence identity between the two proteins. Interactions of ORF158L with the histone H3/H4 complex and H3 were demonstrated by isothermal titration calorimetry (ITC) experiments. Subsequently, the results of ITC studies on structure-based mutants of ORF158L suggested Arg67 and Ala93 were key residues for histone H3 interactions. Moreover, a combination of approaches of ORF158L knockdown and isobaric tags/mass spectrometry for relative and absolute quantifications (iTRAQ) revealed that ORF158L may be involved in both the regulation and the expression of histone H3 and H3 methylation. Our present studies suggest that ORF158L may function as a histone H3 chaperon, enabling it to control host cellular gene expression and to facilitate viral replication.

  7. Kaposi's sarcoma-associated herpesvirus ORF6 gene is essential in viral lytic replication.

    PubMed

    Peng, Can; Chen, Jungang; Tang, Wei; Liu, Chunlan; Chen, Xulin

    2014-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV. PMID:24911362

  8. Specific antibodies induced by inactivated parapoxvirus ovis potently enhance oxidative burst in canine blood polymorphonuclear leukocytes and monocytes.

    PubMed

    Schütze, Nicole; Raue, Rüdiger; Büttner, Mathias; Köhler, Gabriele; McInnes, Colin J; Alber, Gottfried

    2010-01-01

    We have recently shown that inactivated parapoxvirus ovis (iPPVO) effectively stimulates canine blood phagocytes. However, a potential link between innate and adaptive immunity induced by iPPVO remained open. The objective of this study was to define the effects of repeated iPPVO treatment of dogs to evaluate (i) iPPVO-specific antibody production, and (ii) modulation of iPPVO-induced oxidative burst by anti-iPPVO antibodies. Serum analysis of dogs treated repeatedly with iPPVO (Zylexis) showed transient production of non-neutralising iPPVO-specific IgG. There was a correlation between iPPVO-specific IgG levels and enhanced oxidative burst rates in vitro upon transfer of immune sera. Even four years after Zylexis treatment considerably stronger oxidative burst rates in response to iPPVO were observed in monocytes and PMN, whereas only moderate burst rates were detected in monocytes, but not in PMN, from dogs treated with a placebo. Depletion of serum IgG by protein A-sepharose or by parapoxvirus ovis coupled to sepharose abolished the increase of oxidative burst responses and resulted in burst rates similar to blood leukocytes from control dogs. However, uptake of viral particles was found to be independent of iPPVO-specific IgG and restricted to cells with dendritic and monocytic morphology. These data demonstrate that non-neutralising iPPVO-specific IgG is produced during treatment with Zylexis. Moreover, for the first time the interaction of iPPVO with antibodies is shown to enhance oxidative burst.

  9. Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

    PubMed

    Hirata, Hisae; Yamaji, Yasuyuki; Komatsu, Ken; Kagiwada, Satoshi; Oshima, Kenro; Okano, Yukari; Takahashi, Shuichiro; Ugaki, Masashi; Namba, Shigetou

    2010-09-01

    The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed.

  10. sORFs.org: a repository of small ORFs identified by ribosome profiling.

    PubMed

    Olexiouk, Volodimir; Crappé, Jeroen; Verbruggen, Steven; Verhegen, Kenneth; Martens, Lennart; Menschaert, Gerben

    2016-01-01

    With the advent of ribosome profiling, a next generation sequencing technique providing a "snap-shot'' of translated mRNA in a cell, many short open reading frames (sORFs) with ribosomal activity were identified. Follow-up studies revealed the existence of functional peptides, so-called micropeptides, translated from these 'sORFs', indicating a new class of bio-active peptides. Over the last few years, several micropeptides exhibiting important cellular functions were discovered. However, ribosome occupancy does not necessarily imply an actual function of the translated peptide, leading to the development of various tools assessing the coding potential of sORFs. Here, we introduce sORFs.org (http://www.sorfs.org), a novel database for sORFs identified using ribosome profiling. Starting from ribosome profiling, sORFs.org identifies sORFs, incorporates state-of-the-art tools and metrics and stores results in a public database. Two query interfaces are provided, a default one enabling quick lookup of sORFs and a BioMart interface providing advanced query and export possibilities. At present, sORFs.org harbors 263 354 sORFs that demonstrate ribosome occupancy, originating from three different cell lines: HCT116 (human), E14_mESC (mouse) and S2 (fruit fly). sORFs.org aims to provide an extensive sORFs database accessible to researchers with limited bioinformatics knowledge, thus enabling easy integration into personal projects. PMID:26527729

  11. Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

    PubMed

    Smirnova, Ekaterina; Firth, Andrew E; Miller, W Allen; Scheidecker, Danièle; Brault, Véronique; Reinbold, Catherine; Rakotondrafara, Aurélie M; Chung, Betty Y-W; Ziegler-Graff, Véronique

    2015-05-01

    Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement. PMID:25946037

  12. Adenoviral E4orf3 and E4orf6 Proteins, But Not E1B55K, Increase Killing of Cancer Cells by Radiotherapy in vivo

    SciTech Connect

    Liikanen, Ilkka; Dias, Joao D.; Nokisalmi, Petri; Sloniecka, Marta; Kangasniemi, Lotta; Rajecki, Mari; Dobner, Thomas; Tenhunen, Mikko; Kanerva, Anna; Pesonen, Sari; Ahtiainen, Laura Ph.D.; Hemminki, Akseli

    2010-11-15

    Purpose: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. Methods and Materials: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. Results: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. Conclusions: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.

  13. The Emerging World of Small ORFs.

    PubMed

    Hellens, Roger P; Brown, Chris M; Chisnall, Matthew A W; Waterhouse, Peter M; Macknight, Richard C

    2016-04-01

    Small open reading frames (sORFs) are an often overlooked feature of plant genomes. Initially found in plant viral RNAs and considered an interesting curiosity, an increasing number of these sORFs have been shown to encode functional peptides or play a regulatory role. The recent discovery that many of these sORFs initiate with start codons other than AUG, together with the identification of functional small peptides encoded in supposedly noncoding primary miRNA transcripts (pri-miRs), has drastically increased the number of potentially functional sORFs within the genome. Here we review how advances in technology, notably ribosome profiling (RP) assays, are complementing bioinformatics and proteogenomic methods to provide powerful ways to identify these elusive features of plant genomes, and highlight the regulatory roles sORFs can play.

  14. Putative Promoters Isolated From Infectious Hypodermal and Hematopoietic Necrosis Virus of Shrimp Drive Expression of a Reporter Gene in Bacteria, Insect Cells, Fish Cells, and Shrimp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1 kb with three putative open reading frames (ORFs) namely, the left ORF, middle ORF and the right ORF on the same DNA strand. Whereas the left ORF codes for non-s...

  15. Sequence analysis of ORF IV RTBV isolated from tungro infected Oryza sativa L. cv Ciherang

    NASA Astrophysics Data System (ADS)

    Hastilestari, Bernadetta Rina; Astuti, Dwi; Estiati, Amy; Nugroho, Satya

    2015-09-01

    The Effort to increase rice production is often constrained by pest and disease such as Tungro. The Tungro disease is caused by the joint infection with two dissimilar viruses; a bacil-form-DNA virus, the Rice tungro bacilliform virus(RTBV) and the spherical RNA virus, Rice tungro spherical virus (RTSV) and transmitted by Green leafhopper (Nephotettix virescens). The symptom of disease is caused by the presence of RTBV. The genome of RTBV consists of four Open reading frames (ORFs) which encode functional proteins. Of the four, ORF IV is unique because it exists only in RTBV. The most efficient method of generating disease resistance plants is to look for natural sources of resistance genes in wild or germplasm and then transfer the gene and the accompanying resistance in cultivated crop varieties. The aim of this study is, therefore, to isolate and analyze of 1170 bp gene of ORF 4 of Tungro virus isolated from an Indonesian rice cultivar, Ciherang (Oryza sativa L. cv Indica). DNA sequencing analysis using BLAST showed 94% similarity with the reference sequence gen bank Acc.M65026.1. The comparisons and mutation analysis of DNA sequences were discussed in this research.

  16. The ORF4 protein of porcine circovirus type 2 antagonizes apoptosis by stabilizing the concentration of ferritin heavy chain through physical interaction.

    PubMed

    Lv, Qizhuang; Guo, Kangkang; Zhang, Guangfang; Zhang, Yanming

    2016-07-01

    Porcine circovirus type 2 (PCV2) is the primary aetiological agent of porcine circovirus-associated disease in swine. The mechanism of PCV2 pathogenesis remains largely unknown. A newly identified viral protein of PCV2, ORF4, has been suggested to be involved in virus-induced apoptosis. However, there is still no information regarding the molecular mechanism by which ORF4 regulates apoptosis. In this study, we reveal that a physical interaction between the PCV2 ORF4 protein and ferritin heavy chain (FHC) in the cytoplasm of host cells reduced the cellular concentration of FHC. The ORF4-mediated reduction of FHC inhibited reactive oxygen species accumulation in PCV2-infected cells. Consequently, the ORF4 protein inhibited apoptosis in host cells. This may be the first report to describe the mechanism of ORF4 cytoprotection against apoptosis during the early stages of PCV2 infection. PMID:27030984

  17. A 65-Year-Old Female from Connecticut with Orf Infection.

    PubMed

    Estela Cubells, Jose Ramón; Braverman, Irwin; Kashgarian, Michael; Lazova, Rossitza

    2016-01-01

    The virus, which causes orf and induces acute pustular skin lesions in sheep and goats, is transmissible to humans yet is rarely observed in North America. We present a case of a 65-year-old female farmer from Connecticut who contracted orf from her sheep. The clinical and histopathologic features, important to arrive at the correct diagnosis of this uncommon yet important infection, are described. We also discuss the benign nature of this condition and emphasize that treatment is not required. PMID:27504446

  18. A 65-Year-Old Female from Connecticut with Orf Infection

    PubMed Central

    Estela Cubells, Jose Ramón; Braverman, Irwin; Kashgarian, Michael; Lazova, Rossitza

    2016-01-01

    The virus, which causes orf and induces acute pustular skin lesions in sheep and goats, is transmissible to humans yet is rarely observed in North America. We present a case of a 65-year-old female farmer from Connecticut who contracted orf from her sheep. The clinical and histopathologic features, important to arrive at the correct diagnosis of this uncommon yet important infection, are described. We also discuss the benign nature of this condition and emphasize that treatment is not required. PMID:27504446

  19. The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation

    PubMed Central

    Bridges, Rebecca G.; Sohn, Sook-Young; Wright, Jordan

    2016-01-01

    ABSTRACT Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection. PMID:26814176

  20. Kaposi's sarcoma-associated herpesvirus ORF57 functions as a viral splicing factor and promotes expression of intron-containing viral lytic genes in spliceosome-mediated RNA splicing.

    PubMed

    Majerciak, Vladimir; Yamanegi, Koji; Allemand, Eric; Kruhlak, Michael; Krainer, Adrian R; Zheng, Zhi-Ming

    2008-03-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 facilitates the expression of both intronless viral ORF59 genes and intron-containing viral K8 and K8.1 genes (V. Majerciak, N. Pripuzova, J. P. McCoy, S. J. Gao, and Z. M. Zheng, J. Virol. 81:1062-1071, 2007). In this study, we showed that disruption of ORF57 in a KSHV genome led to increased accumulation of ORF50 and K8 pre-mRNAs and reduced expression of ORF50 and K-bZIP proteins but had no effect on latency-associated nuclear antigen (LANA). Cotransfection of ORF57 and K8beta cDNA, which retains a suboptimal intron of K8 pre-mRNA due to alternative splicing, promoted RNA splicing of K8beta and production of K8alpha (K-bZIP). Although Epstein-Barr virus EB2, a closely related homolog of ORF57, had a similar activity in the cotransfection assays, herpes simplex virus type 1 ICP27 was inactive. This enhancement of RNA splicing by ORF57 correlates with the intact N-terminal nuclear localization signal motifs of ORF57 and takes place in the absence of other viral proteins. In activated KSHV-infected B cells, KSHV ORF57 partially colocalizes with splicing factors in nuclear speckles and assembles into spliceosomal complexes in association with low-abundance viral ORF50 and K8 pre-mRNAs and essential splicing components. The association of ORF57 with snRNAs occurs by ORF57-Sm protein interaction. We also found that ORF57 binds K8beta pre-mRNAs in vitro in the presence of nuclear extracts. Collectively our data indicate that KSHV ORF57 functions as a novel splicing factor in the spliceosome-mediated splicing of viral RNA transcripts.

  1. Efficacy of the paramunity inducer PIND-ORF in the treatment of canine parvovirus infection.

    PubMed

    Proksch, A L; Unterer, S; Truyen, U; Hartmann, K

    2014-11-01

    Canine parvovirus (CPV) infection is a common and severe disease particularly affecting young dogs. The paramunity inducer PIND-ORF is reported to stimulate the innate immune system and, if used as a supplementary medication, might lead to a more rapid improvement in clinical signs in dogs with CPV infection. The aim of this study was to evaluate the efficacy of PIND-ORF in dogs with CPV infection in a prospective, placebo-controlled, double-blinded trial using 38 dogs randomly assigned to two groups. Inclusion criteria were clinical signs consistent with CPV infection and a positive faecal CPV PCR. Dogs received either PIND-ORF (n = 20) or placebo (n = 18) and additional symptomatic treatment. Time to recovery and mortality rate were compared between the two groups. Clinical signs, complete blood counts (CBC), and serum protein and albumin concentrations were evaluated daily during hospitalisation and on day 14. Viral shedding and antibody titres were measured by faecal CPV PCR and serum neutralisation assay. There was no significant difference in time to recovery, clinical signs, blood parameters, duration of virus shedding, and antibody titres between the two groups. The only significant difference was an increase in lymphocyte counts and antibody titres observed in the PIND-ORF group only. Three dogs receiving placebo did not survive, but the mortality rate was not significantly different between groups (P = 0.097). No significant effect of PIND-ORF on recovery and outcome could be demonstrated.

  2. Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection

    PubMed Central

    Gaspar, Miguel; Gill, Michael B.; Lösing, Jens-Bernhard; May, Janet S.; Stevenson, Philip G.

    2008-01-01

    All gamma-herpesviruses encode at least one homolog of the cellular enzyme formyl-glycineamide-phosphoribosyl-amidotransferase. Murid herpesvirus-4 (MuHV-4) encodes 3 (ORFs 75a, 75b and 75c), suggesting that at least some copies have acquired new functions. Here we show that the corresponding proteins are all present in virions and localize to infected cell nuclei. Despite these common features, ORFs 75a and 75b did not substitute functionally for a lack of ORF75c, as ORF75c virus knockouts were severely impaired for lytic replication in vitro and for host colonization in vivo. They showed 2 defects: incoming capsids failed to migrate to the nuclear margin following membrane fusion, and genomes that did reach the nucleus failed to initiate normal gene expression. The latter defect was associated with a failure of in-coming virions to disassemble PML bodies. The capsid transport deficit seemed to be functionally more important, since ORF75c− MuHV-4 infected both PML+ and PML− cells poorly. The original host enzyme has therefore evolved into a set of distinct and multi-functional viral tegument proteins. One important function is moving incoming capsids to the nuclear margin for viral genome delivery. PMID:18648660

  3. Mutation of Herpesvirus Saimiri ORF51 Glycoprotein Specifically Targets Infectivity to Hepatocellular Carcinoma Cell Lines

    PubMed Central

    Turrell, Susan J.; Whitehouse, Adrian

    2011-01-01

    Herpesvirus saimiri (HVS) is a gamma herpesvirus with several properties that make it an amenable gene therapy vector; namely its large packaging capacity, its ability to persist as a nonintegrated episome, and its ability to infect numerous human cell types. We used RecA-mediated recombination to develop an HVS vector with a mutated virion protein. The heparan sulphate-binding region of HVS ORF51 was substituted for a peptide sequence which interacts with somatostatin receptors (SSTRs), overexpressed on hepatocellular carcinoma (HCC) cells. HVS mORF51 showed reduced infectivity in non-HCC human cell lines compared to wild-type virus. Strikingly, HVS mORF51 retained its ability to infect HCC cell lines efficiently. However, neutralisation assays suggest that HVS mORF51 has no enhanced binding to SSTRs. Therefore, mutation of the ORF51 glycoprotein has specifically targeted HVS to HCC cell lines by reducing the infectivity of other cell types; however, the mechanism for this targeting is unknown. PMID:21197456

  4. Binding of cellular export factor REF/Aly by Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is not required for efficient KSHV lytic replication.

    PubMed

    Li, Da-Jiang; Verma, Dinesh; Swaminathan, Sankar

    2012-09-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.

  5. Adenovirus E4-ORF3-dependent relocalization of TIF1{alpha} and TIF1{gamma} relies on access to the Coiled-Coil motif

    SciTech Connect

    Vink, Elizabeth I.; Yondola, Mark A.; Wu, Kai; Hearing, Patrick

    2012-01-20

    The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1{alpha}), but not homologous TIF1{beta}. Here, we introduce TIF1{gamma} as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1{gamma} in virus-infected cells in vivo and binds to TIF1{gamma} in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1{alpha}, TIF1{beta}, and TIF1{gamma}, and that the C-terminal half of TIF1{beta} interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

  6. No evidence for translation of pog, a predicted overlapping gene of Solenopsis invicta virus 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An overlapping open reading frame (ORF) with a potential to encode a functional protein has been identified within the 3'-proximal ORF of Solenopsis invicta virus 1 (SINV-1) and three bee viruses. This ORF has been referred to as predicted overlapping gene (pog). Protein motif searches of pog reve...

  7. Characterization of Nora Virus Structural Proteins via Western Blot Analysis

    PubMed Central

    Ericson, Brad L.; Carlson, Darby J.

    2016-01-01

    Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses. PMID:27298753

  8. Molecular characterization of Hop latent virus and phylogenetic relationships among viruses closely related to carlaviruses.

    PubMed

    Hataya, T; Uchino, K; Arimoto, R; Suda, N; Sano, T; Shikata, E; Uyeda, I

    2000-01-01

    The complete nucleotide sequence of the hop latent virus (HpLV) genome was determined. The viral RNA genome is 8,612 nucleotides long, excluding the poly(A) tail, and contains six open reading frames (ORFs), which encode putative proteins of 224-kDa (ORF 1), 25-kDa (ORF 2), 11-kDa (ORF 3), 7-kDa (ORF 4), 34-kDa (ORF 5), and 12-kDa (ORF 6). ORF 5 encodes the coat protein as demonstrated by N-terminal sequencing of three proteolytic peptides derived from the virus particle. The genome organization of HpLV is similar to that of other species in the genus Carlavirus, and the overall sequence of HpLV is more similar to that of Potato virus M than to sequences of other carlaviruses reported to date. The amino acid sequences of the putative methyltransferase, RNA helicase, and RNA-dependent RNA polymerase encoded in ORF 1 and an 'accessory' helicase encoded in ORF 2 of the HpLV genome were compared with those of viruses in the 'tymo' lineage: the genera Carlavirus, Potexvirus, Allexivirus, Foveavirus, Trichovirus, Capillovirus, Vitivirus, and Tymovirus. The phylogenetic relationships among the viruses in these genera are discussed. This is the first molecular characterization of a carlavirus infecting hop plants. PMID:11205102

  9. The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

    PubMed Central

    Kechker, Peter; Sharf, Rakefet; Kleinberger, Tamar

    2016-01-01

    The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition

  10. The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response.

    PubMed

    Brestovitsky, Anna; Nebenzahl-Sharon, Keren; Kechker, Peter; Sharf, Rakefet; Kleinberger, Tamar

    2016-02-01

    The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition

  11. An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function

    PubMed Central

    Orlando, Joseph S.; Ornelles, David A.

    1999-01-01

    A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection. PMID:10233919

  12. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  13. Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

    PubMed

    Griffin, Bryan D; Nagy, Eva

    2011-06-01

    Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

  14. Protein kinase CK2 phosphorylation regulates the interaction of Kaposi's sarcoma-associated herpesvirus regulatory protein ORF57 with its multifunctional partner hnRNP K

    PubMed Central

    Malik, Poonam; Clements, J. Barklie

    2004-01-01

    ORF57 protein of Kaposi's sarcoma-associated herpesvirus has a counterpart in all herpesvirus of mammals and birds and regulates gene expression at transcriptional and post-transcriptional levels. ORF57 was capable of self-interaction and bound a rapidly migrating form of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional cellular protein involved in gene expression. In virus infected cell extracts, ORF57 was present in a complex with hnRNP K that had protein kinase CK2 activity, and was phosphorylated by CK2. Different regions of ORF57 bound both catalytic α/α′ and regulatory β subunits of CK2. CK2 modification enhanced the ORF57–hnRNP K interaction, and may regulate the presence and activities of components in the complex. We suggest that ORF57 and hnRNP K interaction may modulate ORF57-mediated regulation of viral gene expression. Herpesviral ORF57 (Rhadinovirus) and ICP27 (Simplexvirus) proteins both interact with hnRNP K and CK2 implying that adaptation of the ancestral hnRNP K and CK2 to associate with viral regulatory ancestor protein likely pre-dates divergence of these Herpesviridae genera that occurred 200 million years ago. PMID:15486205

  15. The importance of L1 ORF2p cryptic sequence to ORF2p fragment-mediated cytotoxicity.

    PubMed

    Christian, Claiborne M; Kines, Kristine J; Belancio, Victoria P

    2016-01-01

    The Long Interspersed Element 1 (LINE1 or L1) ORF2 protein (ORF2p) can cause DNA damage through the activity of its endonuclease domain (EN). The DNA double-strand breaks (DSB) introduced by the ORF2p EN have the potential to be mutagenic. Previously, our lab has shown that ORF2p fragments containing the EN domain could be expressed in mammalian cells and have variable cytotoxicity. Inclusion of the ORF2p sequence C-terminal to the EN domain in these fragments both reduced the cytotoxicity of these fragments and increased their presence in the nucleus as detected by Western blot analysis. Here, we identify the amino acids (aa 270-274) in the newly-identified ORF2p Cryptic region (Cry) that may be important to the subcellular localization and cytotoxic potential of these EN-containing ORF2p fragments. PMID:27583184

  16. KSHV ORF57, a protein of many faces.

    PubMed

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2015-02-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein (also known as mRNA transcript accumulation (Mta)) is a potent posttranscriptional regulator essential for the efficient expression of KSHV lytic genes and productive KSHV replication. ORF57 possesses numerous activities that promote the expression of viral genes, including the three major functions of enhancement of RNA stability, promotion of RNA splicing, and stimulation of protein translation. The multifunctional nature of ORF57 is driven by its ability to interact with an array of cellular cofactors. These interactions are required for the formation of ORF57-containing ribonucleoprotein complexes at specific binding sites in the target transcripts, referred as Mta-responsive elements (MREs). Understanding of the ORF57 protein conformation has led to the identification of two structurally-distinct domains within the ORF57 polypeptide: an unstructured intrinsically disordered N-terminal domain and a structured α-helix-rich C-terminal domain. The distinct structures of the domains serve as the foundation for their unique binding affinities: the N-terminal domain mediates ORF57 interactions with cellular cofactors and target RNAs, and the C-terminal domain mediates ORF57 homodimerization. In addition, each domain has been found to contribute to the stability of ORF57 protein in infected cells by counteracting caspase- and proteasome-mediated degradation pathways. Together, these new findings provide insight into the function and biological properties of ORF57 in the KSHV life cycle and pathogenesis. PMID:25674768

  17. KSHV ORF57, a Protein of Many Faces

    PubMed Central

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 protein (also known as mRNA transcript accumulation (Mta)) is a potent posttranscriptional regulator essential for the efficient expression of KSHV lytic genes and productive KSHV replication. ORF57 possesses numerous activities that promote the expression of viral genes, including the three major functions of enhancement of RNA stability, promotion of RNA splicing, and stimulation of protein translation. The multifunctional nature of ORF57 is driven by its ability to interact with an array of cellular cofactors. These interactions are required for the formation of ORF57-containing ribonucleoprotein complexes at specific binding sites in the target transcripts, referred as Mta-responsive elements (MREs). Understanding of the ORF57 protein conformation has led to the identification of two structurally-distinct domains within the ORF57 polypeptide: an unstructured intrinsically disordered N-terminal domain and a structured α-helix-rich C-terminal domain. The distinct structures of the domains serve as the foundation for their unique binding affinities: the N-terminal domain mediates ORF57 interactions with cellular cofactors and target RNAs, and the C-terminal domain mediates ORF57 homodimerization. In addition, each domain has been found to contribute to the stability of ORF57 protein in infected cells by counteracting caspase- and proteasome-mediated degradation pathways. Together, these new findings provide insight into the function and biological properties of ORF57 in the KSHV life cycle and pathogenesis. PMID:25674768

  18. LINE-1 ORF1 protein enhances Alu SINE retrotransposition.

    PubMed

    Wallace, Nicholas; Wagstaff, Bradley J; Deininger, Prescott L; Roy-Engel, Astrid M

    2008-08-01

    Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

  19. Severe Acute Respiratory Syndrome Coronavirus ORF7a Inhibits Bone Marrow Stromal Antigen 2 Virion Tethering through a Novel Mechanism of Glycosylation Interference

    PubMed Central

    Taylor, Justin K.; Coleman, Christopher M.; Postel, Sandra; Sisk, Jeanne M.; Bernbaum, John G.; Venkataraman, Thiagarajan; Sundberg, Eric J.

    2015-01-01

    ABSTRACT Severe acute respiratory syndrome (SARS) emerged in November 2002 as a case of atypical pneumonia in China, and the causative agent of SARS was identified to be a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was initially identified to be a pre-B-cell growth promoter, but it also inhibits the release of virions of the retrovirus human immunodeficiency virus type 1 (HIV-1) by tethering budding virions to the host cell membrane. Further work has shown that BST-2 restricts the release of many other viruses, including the human coronavirus 229E (hCoV-229E), and the genomes of many of these viruses encode BST-2 antagonists to overcome BST-2 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has the ability to restrict SARS-CoV and if the SARS-CoV genome encodes any proteins that modulate BST-2's antiviral function. Through an in vitro screen, we identified four potential BST-2 modulators encoded by the SARS-CoV genome: the papain-like protease (PLPro), nonstructural protein 1 (nsp1), ORF6, and ORF7a. As the function of ORF7a in SARS-CoV replication was previously unknown, we focused our study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and found that ORF7a localization changes when BST-2 is overexpressed and ORF7a binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the restriction activity of BST-2 by blocking the glycosylation of BST-2. IMPORTANCE The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from zoonotic sources in 2002 and caused over 8,000 infections and 800 deaths in 37 countries around the world. Identifying host factors that regulate SARS-CoV pathogenesis is critical to understanding how

  20. Middle East respiratory syndrome coronavirus ORF4b protein inhibits type I interferon production through both cytoplasmic and nuclear targets

    PubMed Central

    Yang, Yang; Ye, Fei; Zhu, Na; Wang, Wenling; Deng, Yao; Zhao, Zhengdong; Tan, Wenjie

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel and highly pathogenic human coronavirus and has quickly spread to other countries in the Middle East, Europe, North Africa and Asia since 2012. Previous studies have shown that MERS-CoV ORF4b antagonizes the early antiviral alpha/beta interferon (IFN-α/β) response, which may significantly contribute to MERS-CoV pathogenesis; however, the underlying mechanism is poorly understood. Here, we found that ORF4b in the cytoplasm could specifically bind to TANK binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), suppress the molecular interaction between mitochondrial antiviral signaling protein (MAVS) and IKKε, and inhibit IFN regulatory factor 3 (IRF3) phosphorylation and subsequent IFN-β production. Further analysis showed that ORF4b could also inhibit IRF3 and IRF7-induced production of IFN-β, whereas deletion of the nuclear localization signal of ORF4b abrogated its ability to inhibit IRF3 and IRF7-induced production of IFN-β, but not IFN-β production induced by RIG-I, MDA5, MAVS, IKKε, and TBK-1, suggesting that ORF4b could inhibit the induction of IFN-β in both the cytoplasm and nucleus. Collectively, these results indicate that MERS-CoV ORF4b inhibits the induction of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. Viruses have evolved multiple strategies to evade or thwart a host’s antiviral responses. A novel human coronavirus (HCoV), Middle East respiratory syndrome coronavirus (MERS-CoV), is distinguished from other coronaviruses by its high pathogenicity and mortality. However, virulence determinants that distinguish MERS-CoV from other HCoVs have yet to be identified. MERS-CoV ORF4b antagonizes the early antiviral response, which may contribute to MERS-CoV pathogenesis. Here, we report the identification of the interferon (IFN) antagonism mechanism of MERS-CoV ORF4b. MERS-CoV ORF4b inhibits the production

  1. Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization ▿

    PubMed Central

    Taylor, Adam; Jackson, Brian R.; Noerenberg, Marko; Hughes, David J.; Boyne, James R.; Verow, Mark; Harris, Mark; Whitehouse, Adrian

    2011-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is essential for virus lytic replication. ORF57 regulates virus gene expression at multiple levels, enhancing transcription, stability, nuclear export, and translation of viral transcripts. To enhance the nuclear export of viral intronless transcripts, ORF57 (i) binds viral intronless mRNAs, (ii) shuttles between the nucleus, nucleolus, and the cytoplasm, and (iii) interacts with multiple cellular nuclear export proteins to access the TAP-mediated nuclear export pathway. We investigated the implications on the subcellular trafficking, cellular nuclear export factor recruitment, and ultimately nuclear mRNA export of an ORF57 protein unable to bind RNA. We observed that mutation of a carboxy-terminal RGG motif, which prevents RNA binding, affects the subcellular localization and nuclear trafficking of the ORF57 protein, suggesting that it forms subnuclear aggregates. Further analysis of the mutant shows that although it still retains the ability to interact with cellular nuclear export proteins, it is unable to export viral intronless mRNAs from the nucleus. Moreover, computational molecular modeling and biochemical studies suggest that, unlike the wild-type protein, this mutant is unable to self-associate. Therefore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nuclear trafficking, and multimerization. PMID:21593148

  2. New Genome Sequences of Gamboa Viruses (Family Bunyaviridae, Genus Orthobunyavirus) Isolated in Panama and Argentina

    PubMed Central

    de Lima, Clayton P. S.; Martins, Lívia C.; Aragão Dias, Amarílis; Cardoso, Jedson F.; Silva, Sandro P.; Da Silva, Daisy E. A.; Oliveira, Layanna F.; Vasconcelos, Janaina M.; Ferreira, João Paulo C.; Travassos da Rosa, Amelia P. A.; Guzman, Hilda; Tesh, Robert B.; Vasconcelos, Pedro F. C.

    2014-01-01

    We describe here the nearly complete open reading frame (ORF) of five Gamboa virus strains isolated in Panama and Argentina. The viruses with complete ORF showed the regular genome organization observed in other orthobunyaviruses with exception to the presence of NSs protein. All predicted proteins showed homology with viruses belonging to members of the family Bunyaviridae. PMID:25414487

  3. Discovery and characterization of smORF-encoded bioactive polypeptides.

    PubMed

    Saghatelian, Alan; Couso, Juan Pablo

    2015-12-01

    Analysis of genomes, transcriptomes and proteomes reveals the existence of hundreds to thousands of translated, yet non-annotated, short open reading frames (small ORFs or smORFs). The discovery of smORFs and their protein products, smORF-encoded polypeptides (SEPs), points to a fundamental gap in our knowledge of protein-coding genes. Various studies have identified central roles for smORFs in metabolism, apoptosis and development. The discovery of these bioactive SEPs emphasizes the functional potential of this unexplored class of biomolecules. Here, we provide an overview of this emerging field and highlight the opportunities for chemical biology to answer fundamental questions about these novel genes. Such studies will provide new insights into the protein-coding potential of genomes and identify functional genes with roles in biology and disease. PMID:26575237

  4. Multiple Regions of Kaposi’s Sarcoma-Associated Herpesvirus ORF59 RNA are Required for Its Expression Mediated by Viral ORF57 and Cellular RBM15

    PubMed Central

    Massimelli, Maria Julia; Majerciak, Vladimir; Kang, Jeong-Gu; Liewehr, David J.; Steinberg, Seth M.; Zheng, Zhi-Ming

    2015-01-01

    KSHV ORF57 (MTA) promotes RNA stability of ORF59, a viral DNA polymerase processivity factor. Here, we show that the integrity of both ORF59 RNA ends is necessary for ORF57-mediated ORF59 expression and deletion of both 5’ and 3’ regions, or one end region with a central region, of ORF59 RNA prevents ORF57-mediated translation of ORF59. The ORF59 sequence between nt 96633 and 96559 resembles other known MTA-responsive elements (MREs). ORF57 specifically binds to a stem-loop region from nt 96596–96572 of the MRE, which also binds cellular RBM15. Internal deletion of the MRE from ORF59 led to poor export, but accumulation of nuclear ORF59 RNA in the presence of ORF57 or RBM15. Despite of being translatable in the presence of ORF57, this deletion mutant exhibits translational defect in the presence of RBM15. Together, our results provide novel insight into the roles of ORF57 and RBM15 in ORF59 RNA accumulation and protein translation. PMID:25690794

  5. Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

    PubMed

    Stewart, Meredith; Hardy, Alexandra; Barry, Gerald; Pinto, Rute Maria; Caporale, Marco; Melzi, Eleonora; Hughes, Joseph; Taggart, Aislynn; Janowicz, Anna; Varela, Mariana; Ratinier, Maxime; Palmarini, Massimo

    2015-11-01

    Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50-59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF. PMID:26290332

  6. Characterization of a second open reading frame in genome segment 10 of bluetongue virus

    PubMed Central

    Stewart, Meredith; Hardy, Alexandra; Barry, Gerald; Pinto, Rute Maria; Caporale, Marco; Melzi, Eleonora; Hughes, Joseph; Taggart, Aislynn; Janowicz, Anna; Varela, Mariana

    2015-01-01

    Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50–59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF. PMID:26290332

  7. Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

    PubMed

    Stewart, Meredith; Hardy, Alexandra; Barry, Gerald; Pinto, Rute Maria; Caporale, Marco; Melzi, Eleonora; Hughes, Joseph; Taggart, Aislynn; Janowicz, Anna; Varela, Mariana; Ratinier, Maxime; Palmarini, Massimo

    2015-11-01

    Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50-59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.

  8. Ovine herpesvirus-2-encoded microRNAs target virus genes involved in virus latency.

    PubMed

    Riaz, Aayesha; Dry, Inga; Levy, Claire S; Hopkins, John; Grey, Finn; Shaw, Darren J; Dalziel, Robert G

    2014-02-01

    Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5' UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3' UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis. PMID:24172907

  9. Molecular and virological studies on contagious pustular dermatitis isolates from Egyptian sheep and goats.

    PubMed

    Mahmoud, Mohamed; Abdelrahman, Khaled; Soliman, Hatem

    2010-10-01

    Orf virus was clinically diagnosed from different field cases of sheep and goat in Hawamdia, Giza, Egypt during the summer 2006. Skin scabs were collected and used for virus isolation, electron microscopy, PCR and sequencing for confirmation, and differential diagnosis. The aetiological virus was fruitfully isolated on the chorio-allantoic membrane of SPF embryonated chicken eggs indicated by expressing the characteristic pock lesions of Poxviridae family. Electron microscopy examination exposed negatively stained oval-shape virus particles trait for members of the genus Parapoxvirus. A 392 bp fragment of the late transcription factor (VLTF-1) gene of orf virus was amplified by PCR from the DNA extracted from the isolates. Phylogenetic analysis revealed 99% identity with other orf virus strains reported worldwide. Selection and processing of clinical specimens and PCR assay applied in this endeavor, presented a reliable laboratory diagnostic tool for orf infections and first molecular characterization of Egyptian orf isolates. PMID:20304450

  10. The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses

    PubMed Central

    Hull, R.; Sadler, J.; Longstaff, M.

    1986-01-01

    Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin. PMID:16453731

  11. small ORFs: A new class of essential genes for development

    PubMed Central

    Albuquerque, João Paulo; Tobias-Santos, Vitória; Rodrigues, Aline Cáceres; Mury, Flávia Borges; da Fonseca, Rodrigo Nunes

    2015-01-01

    Genes that contain small open reading frames (smORFs) constitute a new group of eukaryotic genes and are expected to represent 5% of the Drosophila melanogaster transcribed genes. In this review we provide a historical perspective of their recent discovery, describe their general mechanism and discuss the importance of smORFs for future genomic and transcriptomic studies. Finally, we discuss the biological role of the most studied smORF so far, the Mlpt/Pri/Tal gene in arthropods. The pleiotropic action of Mlpt/Pri/Tal in D. melanogaster suggests a complex evolutionary scenario that can be used to understand the origins, evolution and integration of smORFs into complex gene regulatory networks. PMID:26500431

  12. Complete nucleotide sequence of rose yellow leaf virus, a new member of the family Tombusviridae.

    PubMed

    Mollov, Dimitre; Lockhart, Ben; Zlesak, David C

    2014-10-01

    The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae. PMID:24838852

  13. A Bovine Herpesvirus 1 Protein Expressed in Latently Infected Neurons (ORF2) Promotes Neurite Sprouting in the Presence of Activated Notch1 or Notch3

    PubMed Central

    Sinani, Devis; Frizzo da Silva, Leticia

    2013-01-01

    Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can lead to life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia, but stress can induce reactivation from latency. The latency-related (LR) RNA is the only viral transcript abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) is not reactivated from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays a crucial role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and inhibits its ability to trans activate certain viral promoters. Notch3 RNA and protein levels are increased during reactivation from latency, suggesting that Notch may promote reactivation. Activated Notch signaling interferes with neuronal differentiation, in part because neurite and axon generation is blocked. In this study, we demonstrated that ORF2 promotes neurite formation in mouse neuroblastoma cells overexpressing Notch1 or Notch3. ORF2 also interfered with Notch-mediated trans activation of the promoter that regulates the expression of Hairy Enhancer of Split 5, an inhibitor of neurite formation. Additional studies provided evidence that ORF2 promotes the degradation of Notch3, but not that of Notch1, in a proteasome-dependent manner. In summary, these studies suggest that ORF2 promotes a mature neuronal phenotype that enhances the survival of infected neurons and consequently increases the pool of latently infected neurons. PMID:23152506

  14. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination

    PubMed Central

    Lau, Susanna K. P.; Feng, Yun; Chen, Honglin; Luk, Hayes K. H.; Yang, Wei-Hong; Li, Kenneth S. M.; Zhang, Yu-Zhen; Huang, Yi; Song, Zhi-Zhong; Chow, Wang-Ngai; Fan, Rachel Y. Y.; Ahmed, Syed Shakeel; Yeung, Hazel C.; Lam, Carol S. F.; Cai, Jian-Piao; Wong, Samson S. Y.; Chan, Jasper F. W.; Yuen, Kwok-Yung

    2015-01-01

    -CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission. IMPORTANCE Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited <40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination. PMID:26269185

  15. ZAP inhibits murine gammaherpesvirus 68 ORF64 expression and is antagonized by RTA.

    PubMed

    Xuan, Yifang; Gong, Danyang; Qi, Jing; Han, Chuanhui; Deng, Hongyu; Gao, Guangxia

    2013-03-01

    Zinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity. PMID:23255809

  16. Kaposi΄s sarcoma-associated herpesvirus ORF36 protein induces chromosome condensation and phosphorylation of histone H3.

    PubMed

    Kim, Sunmi; Cha, Seho; Jang, Jun Hyeong; Kim, Yejin; Seo, Taegun

    2013-01-01

    Kaposi΄s sarcoma-associated herpesvirus (KSHV) has been known as an agent causing Kaposi΄s sarcoma, primary effusion lymphoma, and multicentric Castleman΄s disease. In the lytic phase of the virus cycle, various viral genes are expressed, which causes host cell dysregulation. Among the lytic genes, viral protein kinase (vPK) encoded by ORF36 is a member of serine/threonine protein kinase (CHPK) family, which is involved in viral gene expression, viral DNA replication and encapsidation, and nuclear egress of virions. Recent studies have shown that the BGLF4 protein of Epstein-Barr virus (EBV), a member of the CHPK family, alters the host cell chromatin structure through phosphorylation of its key regulators. The role of KSHV ORF36 in cellular mitotic events, however, is not yet understood. In the current study, we showed that KSHV ORF36 induced chromosome condensation and phosphorylation of histone H3 on Ser 10, which are known as cellular mitosis markers. These processes have occurred in a kinase activity-dependent manner. PMID:23530827

  17. Crystal Structure of Xanthomonas AvrRxo1-ORF1, a Type III Effector with a Polynucleotide Kinase Domain, and Its Interactor AvrRxo1-ORF2.

    PubMed

    Han, Qian; Zhou, Changhe; Wu, Shuchi; Liu, Yi; Triplett, Lindsay; Miao, Jiamin; Tokuhisa, James; Deblais, Loïc; Robinson, Howard; Leach, Jan E; Li, Jianyong; Zhao, Bingyu

    2015-10-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) disease on rice plants. Xoc delivers a type III effector AvrRxo1-ORF1 into rice plant cells that can be recognized by disease resistance (R) protein Rxo1, and triggers resistance to BLS disease. However, the mechanism and virulence role of AvrRxo1 is not known. In the genome of Xoc, AvrRxo1-ORF1 is adjacent to another gene AvrRxo1-ORF2, which was predicted to encode a molecular chaperone of AvrRxo1-ORF1. We report the co-purification and crystallization of the AvrRxo1-ORF1:AvrRxo1-ORF2 tetramer complex at 1.64 Å resolution. AvrRxo1-ORF1 has a T4 polynucleotide kinase domain, and expression of AvrRxo1-ORF1 suppresses bacterial growth in a manner dependent on the kinase motif. Although AvrRxo1-ORF2 binds AvrRxo1-ORF1, it is structurally different from typical effector-binding chaperones, in that it has a distinct fold containing a novel kinase-binding domain. AvrRxo1-ORF2 functions to suppress the bacteriostatic activity of AvrRxo1-ORF1 in bacterial cells.

  18. Orchid fleck virus is a rhabdovirus with an unusual bipartite genome.

    PubMed

    Kondo, Hideki; Maeda, Takanori; Shirako, Yukio; Tamada, Tetsuo

    2006-08-01

    Orchid fleck virus (OFV) has an unusual bipartite negative-sense RNA genome with clear sequence similarities to those of nucleorhabdoviruses. The OFV genome consists of two single-stranded RNA molecules, RNA1 and RNA2 that are 6413 and 6001 nt long, respectively, with open reading frame (ORF) information in the complementary sense. RNA1 encodes 49 (ORF1), 26 (ORF2), 38 (ORF3), 20 (ORF4) and 61 kDa (ORF5) proteins, and RNA2 encodes a single protein of 212 kDa (ORF6). ORF1, ORF5 and ORF6 proteins had significant similarities (21-38 % identity) to the nucleocapsid protein (N), glycoprotein (G) and polymerase (L) gene products, respectively, of other rhabdoviruses, especially nucleorhabdoviruses, whereas ORF2, ORF3 and ORF4 proteins had no significant similarities to other proteins in the international databases. Similarities between OFV and rhabdoviruses were also found in the sequence complementarity at both termini of each RNA segment (the common terminal sequences are 3'-UGUGUC---GACACA-5'), the conserved intergenic sequences and in being negative sense. It was proposed that a new genus Dichorhabdovirus in the family Rhabdoviridae of the order Mononegavirales should be established with OFV as its prototype member and type species.

  19. Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

    PubMed Central

    Mylona, P V; Premakumar, R; Pau, R N; Bishop, P E

    1996-01-01

    In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions. PMID:8550418

  20. Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

    PubMed

    Mylona, P V; Premakumar, R; Pau, R N; Bishop, P E

    1996-01-01

    In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions.

  1. Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein: Exploiting All Stages of Viral mRNA Processing

    PubMed Central

    Schumann, Sophie; Jackson, Brian R.; Baquero-Perez, Belinda; Whitehouse, Adrian

    2013-01-01

    Nuclear mRNA export is a highly complex and regulated process in cells. Cellular transcripts must undergo successful maturation processes, including splicing, 5'-, and 3'-end processing, which are essential for assembly of an export competent ribonucleoprotein particle. Many viruses replicate in the nucleus of the host cell and require cellular mRNA export factors to efficiently export viral transcripts. However, some viral mRNAs undergo aberrant mRNA processing, thus prompting the viruses to express their own specific mRNA export proteins to facilitate efficient export of viral transcripts and allowing translation in the cytoplasm. This review will focus on the Kaposi’s sarcoma-associated herpesvirus ORF57 protein, a multifunctional protein involved in all stages of viral mRNA processing and that is essential for virus replication. Using the example of ORF57, we will describe cellular bulk mRNA export pathways and highlight their distinct features, before exploring how the virus has evolved to exploit these mechanisms. PMID:23896747

  2. Involvement of Bombyx mori nucleopolyhedrovirus ORF41 (Bm41) in BV production and ODV envelopment

    SciTech Connect

    Tian Caihong; Zhao Jinfang; Xu Yipeng; Xue Jian; Zhang Baoqin; Cui Yingjun; Zhang Minjuan; Bao Yanyuan; Zhang Chuanxi

    2009-04-25

    Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBm{sup De}) and a repaired virus (vBm{sup Re}) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD{sub 50} bioassay and took 18-23 h longer to kill larvae than wild-type virus in an LT{sub 50} bioassay.

  3. C9orf72 expansion presenting as an eating disorder.

    PubMed

    Sanders, Peter; Ewing, Isobel; Ahmad, Kate

    2016-03-01

    This report describes a 64-year-old woman with a strong family history of motor neuron disease, whose diagnosis of behavioural variant frontotemporal dementia was delayed due to her initial presentation with atypical manifestations, including restriction of oral intake resulting in low weight, disordered eating and anxiety. Upon investigation, she was found to be a carrier of the C9orf72 hexanucleotide repeat expansion. Our case supports previous publications asserting that C9orf72 mutation carriers manifest with diverse clinical syndromes, and expands the phenotype to include anorexia and food refusal as potential features of the condition. PMID:26547294

  4. The C9ORF72 repeat expansion disrupts nucleocytoplasmic transport

    PubMed Central

    Haeusler, Aaron R.; Grima, Jonathan C.; Machamer, James B.; Steinwald, Peter; Daley, Elizabeth L.; Miller, Sean J.; Cunningham, Kathleen M.; Vidensky, Svetlana; Gupta, Saksham; Thomas, Michael A.; Hong, Ingie; Chiu, Shu-Ling; Huganir, Richard L.; Ostrow, Lyle W.; Matunis, Michael J.; Wang, Jiou; Sattler, Rita

    2016-01-01

    A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila ortholog of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9ORF72 ALS patient-derived induced pluripotent stem cells (iPSNs), and in C9ORF72 patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9ORF72 iPSNs, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD amenable to pharmacotherapeutic intervention. PMID:26308891

  5. Beyond ORF: Student-Level Predictors of Reading Achievement

    ERIC Educational Resources Information Center

    Canto, Angela I.; Proctor, Briley E.

    2013-01-01

    This study explored student-level predictors of reading achievement among third grade regular education students. Predictors included student demographics (sex and socioeconomic status (SES), using free and reduced lunch as proxy for SES), direct observations of reading skills (oral reading fluency (ORF) and word decoding skill (nonsense word…

  6. Comparative genomic analysis of hyperthermophilic archaeal fuselloviridae viruses

    SciTech Connect

    B. Wiedenheft; K. Stedman; F. Roberto; D. Willits; A. K. Gleske; L. Zoeller; J. Snyder; T. Douglas; M. Young

    2004-02-01

    The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindleshaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of _15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.

  7. The myxoma virus thymidine kinase gene: sequence and transcriptional mapping.

    PubMed

    Jackson, R J; Bults, H G

    1992-02-01

    The myxoma virus thymidine kinase (TK) gene is encoded on a 1.6 kb SacI-SalI restriction fragment located between 57.7 and 59.3 kb on the 163 kb genomic map. The nucleotide sequence of this fragment as well as 228 bp from the adjacent SalI-AA2 fragment was determined and found to encode four major open reading frames (ORFs). Three of these ORFs are similar in nucleotide sequence to ORFs L5R and J1R, and the TK gene of vaccinia virus (VV). The fourth ORF, MF8a, shows similarity to the ORFs found in the same position relative to the TK genes of Shope fibroma virus, Kenya sheep-1 virus and swine-pox virus. A search of the complete VV nucleotide sequence for regions of similarity to MF8a identified the host specificity gene C7L. Northern blot analysis of early viral RNA identified transcripts of approximately 700 nucleotides for both the TK gene and ORF MF8a. The 5' ends of the TK gene and ORF MF8a early mRNAs were mapped by primer extension to initiation sites 13 nucleotides downstream of sequences with similarity to the VV early promoter consensus. The sizes of the TK and MF8a mRNAs are consistent with transcription termination and polyadenylation occurring downstream of the sequence TTTTTNT, which is identical to the consensus sequence for the VV transcription termination signal.

  8. Complete genome sequence of motherwort yellow mottle virus, a novel putative member of the genus Torradovirus.

    PubMed

    Seo, Jang-Kyun; Kang, Minji; Kwak, Hae-Ryun; Kim, Mi-Kyeong; Kim, Chang-Seok; Lee, Su-Heon; Kim, Jeong-Soo; Choi, Hong-Soo

    2015-02-01

    The complete genome sequence of a new virus isolated from a motherwort plant exhibiting yellow mottle, mild mosaic, and stunting symptoms in Andong, Korea, was determined. The genome of this virus is composed of two single-stranded RNAs (7068 and 4963 nucleotides in length, respectively) carrying poly(A) tails. RNA1 contains one large open reading frame (RNA1-ORF1), while two potential ORFs (RNA2-ORF1 and RNA2-ORF2) were found in RNA2. BLAST searches of protein databases showed that RNA1-ORF1 and RNA2-ORF2 have maximum amino acid sequence identities of 53 % and 57 % to the RNA1-ORF1 and RNA2-ORF2, respectively, of lettuce necrotic leaf curl virus (LNLCV, a recently identified torradovirus). Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Torradovirus. The name "motherwort yellow mottle virus" (MYMoV) is proposed for this new virus.

  9. A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing

    PubMed Central

    Untiveros, Milton; Olspert, Allan; Artola, Katrin

    2016-01-01

    Summary The single‐stranded, positive‐sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3′ third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the ‘transframe’ product, P1N‐PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N‐PISPO inhibited short‐distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co‐opted for the evolution and expression of further novel gene products. PMID:26757490

  10. Immunological and biochemical characterisation of 7ap, a short protein translated from an alternative frame of ORF7 of PRRSV.

    PubMed

    Olasz, Ferenc; Dénes, Béla; Bálint, Ádám; Magyar, Tibor; Belák, Sándor; Zádori, Zoltán

    2016-06-01

    Sequence analysis revealed a short alternative open reading frame (ORF) named ORF7a within the nucleocapsid gene of genetically divergent porcine reproductive and respiratory syndrome virus (PRRSV) genomes. Alignment of the corresponding protein sequences (named 7ap) revealed substantial heterogeneity among 7aps of different genotypes, though all of them are predicted to be positively charged. Green fluorescent protein and FLAG fusion constructs of ORF7a of the HU-14432/2011 PRRSV demonstrated that 7ap is expressed. 7ap of HU- 14432/2011 (Hu7ap) was synthesised chemically, and ELISA experiments revealed that Hu7ap binds strongly to mammalian IgGs. Protein-protein gel retardation assays and complement fixation inhibition suggest that 7aps bind to the CH2 domain of the IgG(Fc) fragment. Cellular localisation and immunological characteristics of PRRSV 7ap may indicate multiple functions including nuclear and cytoplasmic over-tuning of normal cellular processes and immunosuppression. PMID:27342098

  11. Mapping and Serodiagnostic Application of a Dominant Epitope within the Human Herpesvirus 8 ORF 65-Encoded Protein

    PubMed Central

    Pau, Chou-Pong; Lam, Lee L.; Spira, Thomas J.; Black, Jodi B.; Stewart, John A.; Pellett, Philip E.; Respess, Richard A.

    1998-01-01

    A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi’s sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8. PMID:9620379

  12. Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas.

    PubMed

    Renault, Tristan; Moreau, Pierrick; Faury, Nicole; Pepin, Jean-François; Segarra, Amélie; Webb, Stephen

    2012-05-01

    Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 "specimens" collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.

  13. Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and kelp fly virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the stru...

  14. Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

    PubMed Central

    Cvijović, Marija; Dalevi, Daniel; Bilsland, Elizabeth; Kemp, Graham JL; Sunnerhagen, Per

    2007-01-01

    Background The translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis. Results We have used comparative genomics to identify novel uORFs in yeast with a high likelihood of having a translational regulatory role. We examined uORFs, previously shown to play a role in regulation of translation in Saccharomyces cerevisiae, for evolutionary conservation within seven Saccharomyces species. Inspection of the set of conserved uORFs yielded the following three characteristics useful for discrimination of functional from spurious uORFs: a length between 4 and 6 codons, a distance from the start of the main ORF between 50 and 150 nucleotides, and finally a lack of overlap with, and clear separation from, neighbouring uORFs. These derived rules are inherently associated with uORFs with properties similar to the GCN4 locus, and may not detect most uORFs of other types. uORFs with high scores based on these rules showed a much higher evolutionary conservation than randomly selected uORFs. In a genome-wide scan in S. cerevisiae, we found 34 conserved uORFs from 32 genes that we predict to be functional; subsequent analysis showed the majority of these to be located within transcripts. A total of 252 genes were found containing conserved uORFs with properties indicative of a functional role; all but 7 are novel. Functional content analysis of this set identified an overrepresentation of genes involved in transcriptional control and development. Conclusion Evolutionary conservation of uORFs in yeasts can be traced up to 100 million years of separation. The

  15. Interaction of Kaposi's sarcoma-associated herpesvirus ORF59 with oriLyt is dependent on binding with K-Rta.

    PubMed

    Rossetto, Cyprian C; Susilarini, Ni Ketut; Pari, Gregory S

    2011-04-01

    Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) displays two distinct life stages, latency and lytic reactivation. Progression through the lytic cycle and replication of the viral genome constitute an essential step toward the production of infectious virus and human disease. KSHV K-RTA has been shown to be the major transactivator required for the initiation of lytic reactivation. In the transient-cotransfection replication assay, K-Rta is the only noncore protein required for DNA synthesis. K-Rta was shown to interact with both C/EBPα binding motifs and the R response elements (RRE) within oriLyt. It is postulated that K-Rta acts in part to facilitate the recruitment of replication factors to oriLyt. In order to define the role of K-Rta in the initiation of lytic DNA synthesis, we show an interaction with ORF59, the DNA polymerase processivity factor (PF), one of the eight virally encoded proteins necessary for origin-dependent DNA replication. Using the chromatin immunoprecipitation (ChIP) assay, both K-Rta and ORF59 interact with the RRE and C/EBPα binding motifs within oriLyt in cells harboring the KSHV bacterial artificial chromosome (BAC). A transient-transfection ChIP assay demonstrated that the interaction of ORF59 with oriLyt is dependent on binding with K-Rta and that ORF59 fails to bind to oriLyt in the absence of K-Rta. Also, using the cotransfection replication assay, overexpression of the interaction domain of K-Rta with ORF59 has a dominant negative effect on oriLyt amplification, suggesting that the interaction of K-Rta with ORF59 is essential for DNA synthesis and supporting the hypothesis that K-Rta facilitates the formation of a replication complex at oriLyt. PMID:21289111

  16. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    PubMed

    Tunnicliffe, Richard B; Hautbergue, Guillaume M; Kalra, Priti; Jackson, Brian R; Whitehouse, Adrian; Wilson, Stuart A; Golovanov, Alexander P

    2011-01-06

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  17. Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Kalra, Priti; Jackson, Brian R.; Whitehouse, Adrian; Wilson, Stuart A.; Golovanov, Alexander P.

    2011-01-01

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway. PMID:21253573

  18. Inactivation of C4orf26 in toothless placental mammals.

    PubMed

    Springer, Mark S; Starrett, James; Morin, Phillip A; Lanzetti, Agnese; Hayashi, Cheryl; Gatesy, John

    2016-02-01

    Previous studies have reported inactivated copies of six enamel-related genes (AMBN, AMEL, AMTN, ENAM, KLK4, MMP20) and one dentin-related gene (DSPP) in one or more toothless vertebrates and/or vertebrates with enamelless teeth, thereby providing evidence that these genes are enamel or tooth-specific with respect to their critical functions that are maintained by natural selection. Here, we employ available genome sequences for edentulous and enamelless mammals to evaluate the enamel specificity of four genes (WDR72, SLC24A4, FAM83H, C4orf26) that have been implicated in amelogenesis imperfecta, a condition in which proper enamel formation is abrogated during tooth development. Coding sequences for WDR72, SCL24A4, and FAM83H are intact in four edentulous taxa (Chinese pangolin, three baleen whales) and three taxa (aardvark, nine-banded armadillo, Hoffmann's two-toed sloth) with enamelless teeth, suggesting that these genes have critical functions beyond their involvement in tooth development. By contrast, genomic data for C4orf26 reveal inactivating mutations in pangolin and bowhead whale as well as evidence for deletion of this gene in two minke whale species. Hybridization capture of exonic regions and PCR screens provide evidence for inactivation of C4orf26 in eight additional baleen whale species. However, C4orf26 is intact in all three species with enamelless teeth that were surveyed, as well as in 95 additional mammalian species with enamel-capped teeth. Estimates of selection intensity suggest that dN/dS ratios on branches leading to taxa with enamelless teeth are similar to the dN/dS ratio on branches leading to taxa with enamel-capped teeth. Based on these results, we conclude that C4orf26 is tooth-specific, but not enamel-specific, with respect to its essential functions that are maintained by natural selection. A caveat is that an alternative splice site variant, which translates exon 3 in a different reading frame, is putatively functional in

  19. Inactivation of C4orf26 in toothless placental mammals.

    PubMed

    Springer, Mark S; Starrett, James; Morin, Phillip A; Lanzetti, Agnese; Hayashi, Cheryl; Gatesy, John

    2016-02-01

    Previous studies have reported inactivated copies of six enamel-related genes (AMBN, AMEL, AMTN, ENAM, KLK4, MMP20) and one dentin-related gene (DSPP) in one or more toothless vertebrates and/or vertebrates with enamelless teeth, thereby providing evidence that these genes are enamel or tooth-specific with respect to their critical functions that are maintained by natural selection. Here, we employ available genome sequences for edentulous and enamelless mammals to evaluate the enamel specificity of four genes (WDR72, SLC24A4, FAM83H, C4orf26) that have been implicated in amelogenesis imperfecta, a condition in which proper enamel formation is abrogated during tooth development. Coding sequences for WDR72, SCL24A4, and FAM83H are intact in four edentulous taxa (Chinese pangolin, three baleen whales) and three taxa (aardvark, nine-banded armadillo, Hoffmann's two-toed sloth) with enamelless teeth, suggesting that these genes have critical functions beyond their involvement in tooth development. By contrast, genomic data for C4orf26 reveal inactivating mutations in pangolin and bowhead whale as well as evidence for deletion of this gene in two minke whale species. Hybridization capture of exonic regions and PCR screens provide evidence for inactivation of C4orf26 in eight additional baleen whale species. However, C4orf26 is intact in all three species with enamelless teeth that were surveyed, as well as in 95 additional mammalian species with enamel-capped teeth. Estimates of selection intensity suggest that dN/dS ratios on branches leading to taxa with enamelless teeth are similar to the dN/dS ratio on branches leading to taxa with enamel-capped teeth. Based on these results, we conclude that C4orf26 is tooth-specific, but not enamel-specific, with respect to its essential functions that are maintained by natural selection. A caveat is that an alternative splice site variant, which translates exon 3 in a different reading frame, is putatively functional in

  20. Purification of a baculovirus-expressed hepatitis E virus structural protein and utility in an enzyme-linked immunosorbent assay.

    PubMed Central

    He, J; Ching, W M; Yarbough, P; Wang, H; Carl, M

    1995-01-01

    We report on the purification of the full-length structural protein encoded by open reading frame 2 (ORF-2) of hepatitis E virus. The ORF-2 protein, expressed in Sf9 cells by using a recombinant baculovirus vector system, was successfully purified to homogeneity. Gel electrophoresis of the purified ORF-2 protein showed a single polypeptide of 75 kDa by Coomassie blue staining and by Western blot (immunoblot) analysis. We demonstrated that the partially purified ORF-2 protein could be used successfully in a sensitive and specific enzyme-linked immunosorbent assay for the detection of antibodies to hepatitis E virus. PMID:8586723

  1. Roles for λ Orf and Escherichia Coli Reco, Recr and Recf in λ Recombination

    PubMed Central

    Sawitzke, J. A.; Stahl, F. W.

    1997-01-01

    Bacteriophage λ lacking its Red recombination functions requires either its own gene product, Orf, or the product of Escherichia coli's recO, recR and recF genes (RecORF) for efficient recombination in recBC sbcB sbcC mutant cells (the RecF pathway). Phage crosses under conditions of a partial block to DNA replication have revealed the following: (1) In the presence of Orf, RecF pathway recombination is similar to λ Red recombination; (2) Orf is necessary for focusing recombination toward the right end of the chromosome as λ is conventionally drawn; (3) RecORF-mediated RecF pathway recombination is not focused toward the right end of the chromosome, which may indicate that RecORF travels along the DNA; (4) both Orf- and RecORF-mediated RecF pathway recombination are stimulated by DNA replication; and (5) low level recombination in the simultaneous absence of Orf and RecORF may occur by a break-copy mechanism that is not initiated by a double strand break. Models for the roles of Orf and RecO, RecR and RecF in recombination are presented. PMID:9335578

  2. Nucleotide sequence of shallot virus X RNA reveals a 5'-proximal cistron closely related to those of potexviruses and a unique arrangement of the 3'-proximal cistrons.

    PubMed

    Kanyuka, K V; Vishnichenko, V K; Levay, K E; Kondrikov DYu; Ryabov, E V; Zavriev, S K

    1992-10-01

    The 8890 nucleotide RNA sequence of shallot virus X (ShVX), a new virus isolated from shallot, has been determined. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5' to 3' direction) of M(r) 194528 (ORF1), 26333 (ORF2), 11245 (ORF3), 42209 (ORF4), 28486 (ORF5) and 14741 (ORF6). The ORF1 protein was found to be highly homologous to the putative potexvirus RNA replicases; ORF2, -3, -5 and -6 proteins also have analogues among the potex- and/or carlavirus-encoded proteins. ORF3 is followed by an AUG-lacking frame coding for an amino acid sequence homologous to that of the 7K to 8K proteins of the triple gene block of the above-mentioned viruses. The putative ORF4 protein has no reliable homology with proteins in the database. The results obtained testify that, except for the unique 42K protein gene, the ShVX genome combines a number of elements typical of both carla- and potexviruses.

  3. Novel Host-Related Virulence Factors Are Encoded by Squirrelpox Virus, the Main Causative Agent of Epidemic Disease in Red Squirrels in the UK

    PubMed Central

    Kjær, Karina Hansen; Wood, Ann R.; Hughes, Margaret; Martensen, Pia Møller; Radford, Alan D.; Hall, Neil; Chantrey, Julian

    2014-01-01

    Squirrelpox virus (SQPV) shows little evidence for morbidity or mortality in North American grey squirrels (Sciurus carolinensis), in which the virus is endemic. However, more recently the virus has emerged to cause epidemics with high mortality in Eurasian red squirrels (S. vulgaris) in Great Britain, which are now threatened. Here we report the genome sequence of SQPV. Comparison with other Poxviridae revealed a core set of poxvirus genes, the phylogeny of which showed SQPV to be in a new Chordopoxvirus subfamily between the Molluscipoxviruses and Parapoxviruses. A number of SQPV genes were related to virulence, including three major histocomaptibility class I homologs, and one CD47 homolog. In addition, a novel potential virulence factor showing homology to mammalian oligoadenylate synthetase (OAS) was identified. This family of proteins normally causes activation of an endoribonuclease (RNaseL) within infected cells. The putative function of this novel SQPV protein was predicted in silico. PMID:24983354

  4. Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell.

    PubMed

    Zhu, Ruo-Lin; Lei, Xiao-Ying; Ke, Fei; Yuan, Xiu-Ping; Zhang, Qi-Ya

    2011-02-01

    Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size.

  5. ORF57 Overcomes the Detrimental Sequence Bias of Kaposi's Sarcoma-Associated Herpesvirus Lytic Genes

    PubMed Central

    Vogt, Carolin; Hackmann, Christian; Rabner, Alona; Koste, Lars; Santag, Susann; Kati, Semra; Mandel-Gutfreund, Yael; Schulz, Thomas F.

    2015-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple posttranscriptional levels. However, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high AT content. Thus, we optimized the K15 cDNA by raising the frequency of GC nucleotides, yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their AT content and found a correlation between AT-richness and ORF57 dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low AT content and are indeed ORF57 independent. The nucleotide composition of K15 resembles that of HIV gag, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIV gag RNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild-type and sequence-optimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression. IMPORTANCE The mostly intronless genes of KSHV are only expressed in the presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead, we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on

  6. Genetic Complementation and Kinetic Analyses of Rhodobacter capsulatus ORF1696 Mutants Indicate that the ORF1696 Protein Enhances Assembly of the Light-Harvesting I Complex

    PubMed Central

    Young, C. S.; Reyes, R. C.; Beatty, J. T.

    1998-01-01

    Rhodobacter capsulatus ORF1696 mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the ORF1696 gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (ΔNae) with a disruption located 13 codons before the 3′ end of the gene. A 5′-proximal disruption after the 31st codon of ORF1696 resulted in a mutant strain (ΔMun) with a novel absorption spectrum. The two strains with more 3′ disruptions (ΔStu and ΔNae) were restored nearly to the parental strain phenotype when trans complemented with a plasmid expressing the ORF1696 gene, but ΔMun was not. The absorption spectrum of ΔMun resembled that of a strain which had a polar mutation in ORF1696. We suggest that a rho-dependent transcription termination site exists between the MunI and proximal StuI sites of ORF1696. A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain ΔStu. In contrast, LHI complex decay occurred 1.7-fold faster in the ORF1696 parental strain than in ΔStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed. PMID:9537372

  7. Primate-specific ORF0 contributes to retrotransposon-mediated diversity.

    PubMed

    Denli, Ahmet M; Narvaiza, Iñigo; Kerman, Bilal E; Pena, Monique; Benner, Christopher; Marchetto, Maria C N; Diedrich, Jolene K; Aslanian, Aaron; Ma, Jiao; Moresco, James J; Moore, Lynne; Hunter, Tony; Saghatelian, Alan; Gage, Fred H

    2015-10-22

    LINE-1 retrotransposons are fast-evolving mobile genetic entities that play roles in gene regulation, pathological conditions, and evolution. Here, we show that the primate LINE-1 5'UTR contains a primate-specific open reading frame (ORF) in the antisense orientation that we named ORF0. The gene product of this ORF localizes to promyelocytic leukemia-adjacent nuclear bodies. ORF0 is present in more than 3,000 loci across human and chimpanzee genomes and has a promoter and a conserved strong Kozak sequence that supports translation. By virtue of containing two splice donor sites, ORF0 can also form fusion proteins with proximal exons. ORF0 transcripts are readily detected in induced pluripotent stem (iPS) cells from both primate species. Capped and polyadenylated ORF0 mRNAs are present in the cytoplasm, and endogenous ORF0 peptides are identified upon proteomic analysis. Finally, ORF0 enhances LINE-1 mobility. Taken together, these results suggest a role for ORF0 in retrotransposon-mediated diversity. PMID:26496605

  8. Complete nucleotide sequence and genome organization of Pelargonium flower break virus.

    PubMed

    Rico, P; Hernández, C

    2004-03-01

    The complete nucleotide sequence of Pelargonium flower break virus (PFBV) has been determined. The genomic RNA is 3923 nucleotides (nt) long and contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 27 kDa protein (p27) and terminates with an amber codon which may be read-through into an in-frame p56 ORF to generate a 86 kDa protein (p86) containing the viral RNA dependent-RNA polymerase motifs. Two small ORFs, located in the central part of the viral genome, encode polypeptides of 7 (p7) and 12 kDa (p12), respectively, which are very likely involved in virus movement. Interestingly, p12 presents a leucine zipper motif that has not been previously reported in related proteins. The 3'-proximal ORF encodes a 37 kDa capsid protein (CP). The p12 ORF is in-frame with the p86 ORF and a double read-through protein of 99 kDa (p99) may be produced. Amino acid sequence comparisons revealed that the proteins encoded by ORFs 2, 3 and 4 are more similar to the corresponding gene products of Carnation mottle virus than to those of other carmoviruses, whereas the p27 and the CP show higher identity with the equivalent proteins of Saguaro cactus virus. Phylogenetic analysis conducted with the different viral products confirmed the assignment of PFBV to the genus Carmovirus. PMID:14991450

  9. Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

    PubMed Central

    den Boon, J A; Snijder, E J; Chirnside, E D; de Vries, A A; Horzinek, M C; Spaan, W J

    1991-01-01

    The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily. Images PMID:1851863

  10. Identification of the simian foamy virus transcriptional transactivator gene (taf).

    PubMed Central

    Mergia, A; Shaw, K E; Pratt-Lowe, E; Barry, P A; Luciw, P A

    1991-01-01

    Simian foamy virus type 1 (SFV-1), a member of spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf). Images PMID:1851862

  11. A negative element involved in Kaposi's sarcoma-associated herpesvirus-encoded ORF11 gene expression

    SciTech Connect

    Chen, Lei

    2009-01-01

    The ORF11 of the Kaposi's sarcoma-associated herpesvirus (KSHV) is a lytic viral gene with delayed-early expression kinetics. How the ORF11 gene expression is regulated in the KSHV lytic cascade is largely unknown. Here we report that the deletion of the KSHV viral IL-6 gene from the viral genome leads to deregulated ORF11 gene expression. The KSHV-encoded viral IL-6 protein was found not to be essentially involved in the regulation of ORF11, suggesting a potential transcriptional cis-regulation. A negative element was identified downstream of the ORF11 gene, which suppresses the ORF11 basal promoter activity in a position-independent manner.

  12. The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection.

    PubMed

    Torrent, F; Villena, A; Lee, P A; Fuchs, W; Bergmann, S M; Coll, J M

    2016-10-01

    Recombinantly expressed fragments of the protein encoded by ORF149 (pORF149), a structural protein from the common- and koi-carp-infecting cyprinid herpesvirus-3 (CyHV-3) that was previously shown to be antigenic, were used to obtain evidence that its amino-terminal part contains immunodominant epitopes in fish populations that survived the infection. To obtain such evidence, nonspecific binding of carp serum tetrameric IgM had to be overcome by a novel ELISA protocol (rec2-ELISA). Rec2-ELISA involved pre-adsorption of carp sera with a heterologous recombinant fragment before incubation with pORF149 fragments and detection with anti-carp IgM monoclonal antibodies. Only in this way was it possible to distinguish between sera from uninfected and survivor carp populations. Although IgM from survivors recognised pORF149 fragments to a lesser degree than whole virus, specificity was confirmed by correlation of rec2- and CyHV-3-ELISAs, inhibition of rec2-ELISA by an excess of frgIIORF149, ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgIIORF149. The similarity of IgM-binding profiles between frgIORF149 (amino acid residues 42-629) and frgIIORF149 (42-159) and their reactivities with previously described anti-CyHV-3 monoclonal antibodies confirmed that most pORF149 epitopes were localised in its amino-terminal part. PMID:27383208

  13. The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection.

    PubMed

    Torrent, F; Villena, A; Lee, P A; Fuchs, W; Bergmann, S M; Coll, J M

    2016-10-01

    Recombinantly expressed fragments of the protein encoded by ORF149 (pORF149), a structural protein from the common- and koi-carp-infecting cyprinid herpesvirus-3 (CyHV-3) that was previously shown to be antigenic, were used to obtain evidence that its amino-terminal part contains immunodominant epitopes in fish populations that survived the infection. To obtain such evidence, nonspecific binding of carp serum tetrameric IgM had to be overcome by a novel ELISA protocol (rec2-ELISA). Rec2-ELISA involved pre-adsorption of carp sera with a heterologous recombinant fragment before incubation with pORF149 fragments and detection with anti-carp IgM monoclonal antibodies. Only in this way was it possible to distinguish between sera from uninfected and survivor carp populations. Although IgM from survivors recognised pORF149 fragments to a lesser degree than whole virus, specificity was confirmed by correlation of rec2- and CyHV-3-ELISAs, inhibition of rec2-ELISA by an excess of frgIIORF149, ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgIIORF149. The similarity of IgM-binding profiles between frgIORF149 (amino acid residues 42-629) and frgIIORF149 (42-159) and their reactivities with previously described anti-CyHV-3 monoclonal antibodies confirmed that most pORF149 epitopes were localised in its amino-terminal part.

  14. Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

    PubMed Central

    DeHart, Caroline J.; Perlman, David H.

    2015-01-01

    ABSTRACT Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1–17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076–1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. IMPORTANCE The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3

  15. The complete sequence of soybean chlorotic mottle virus DNA and the identification of a novel promoter.

    PubMed

    Hasegawa, A; Verver, J; Shimada, A; Saito, M; Goldbach, R; Van Kammen, A; Miki, K; Kameya-Iwaki, M; Hibi, T

    1989-12-11

    The complete nucleotide sequence of an infectious clone of soybean chlorotic mottle virus (SoyCMV) DNA was determined and compared with those of three other caulimoviruses, cauliflower mosaic virus (CaMV), carnation etched ring virus and figwort mosaic virus. The double-stranded DNA genome of SoyCMV (8,175 bp) contained nine open reading frames (ORFs) and one large intergenic region. The primer binding sites, gene organization and size of ORFs were similar to those of the other caulimoviruses, except for ORF I, which was split into ORF Ia and Ib. The amino acid sequences deduced from each ORF showed only short, highly homologous regions in several of the corresponding ORFs of the three other caulimoviruses. A promoter fragment of 378 bp in SoyCMV ORF III showed a strong expression activity, comparable to that of the CaMV 35S promoter, in tobacco mesophyll protoplasts as determined by a beta-glucuronidase assay using electrotransfection. The fragment contained CAAT and TATA boxes but no transcriptional enhancer signal as reported for the CaMV 35S promoter. Instead, it had sequences homologous to a part of the translational enhancer signal reported for the 5'-leader sequence of tobacco mosaic virus RNA.

  16. C9orf72 is required for proper macrophage and microglial function in mice.

    PubMed

    O'Rourke, J G; Bogdanik, L; Yáñez, A; Lall, D; Wolf, A J; Muhammad, A K M G; Ho, R; Carmona, S; Vit, J P; Zarrow, J; Kim, K J; Bell, S; Harms, M B; Miller, T M; Dangler, C A; Underhill, D M; Goodridge, H S; Lutz, C M; Baloh, R H

    2016-03-18

    Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers. PMID:26989253

  17. uORFdb—a comprehensive literature database on eukaryotic uORF biology

    PubMed Central

    Wethmar, Klaus; Barbosa-Silva, Adriano; Andrade-Navarro, Miguel A.; Leutz, Achim

    2014-01-01

    Approximately half of all human transcripts contain at least one upstream translational initiation site that precedes the main coding sequence (CDS) and gives rise to an upstream open reading frame (uORF). We generated uORFdb, publicly available at http://cbdm.mdc-berlin.de/tools/uorfdb, to serve as a comprehensive literature database on eukaryotic uORF biology. Upstream ORFs affect downstream translation by interfering with the unrestrained progression of ribosomes across the transcript leader sequence. Although the first uORF-related translational activity was observed >30 years ago, and an increasing number of studies link defective uORF-mediated translational control to the development of human diseases, the features that determine uORF-mediated regulation of downstream translation are not well understood. The uORFdb was manually curated from all uORF-related literature listed at the PubMed database. It categorizes individual publications by a variety of denominators including taxon, gene and type of study. Furthermore, the database can be filtered for multiple structural and functional uORF-related properties to allow convenient and targeted access to the complex field of eukaryotic uORF biology. PMID:24163100

  18. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

    PubMed Central

    Farg, Manal A.; Sundaramoorthy, Vinod; Sultana, Jessica M.; Yang, Shu; Atkinson, Rachel A.K.; Levina, Vita; Halloran, Mark A.; Gleeson, Paul A.; Blair, Ian P.; Soo, Kai Y.; King, Anna E.; Atkin, Julie D.

    2014-01-01

    Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking. PMID:24549040

  19. E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling

    PubMed Central

    Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V.

    2016-01-01

    Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling. PMID:27537838

  20. E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling.

    PubMed

    Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V

    2016-01-01

    Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling. PMID:27537838

  1. Sequence and Transcriptional Analyses of the Fish Retroviruses Walleye Epidermal Hyperplasia Virus Types 1 and 2: Evidence for a Gene Duplication

    PubMed Central

    LaPierre, Lorie A.; Holzschu, Donald L.; Bowser, Paul R.; Casey, James W.

    1999-01-01

    Walleye epidermal hyperplasia virus types 1 and 2 (WEHV1 and WEHV2, respectively) are associated with a hyperproliferative skin lesion on walleyes that appears and regresses seasonally. We have determined the complete nucleotide sequences and transcriptional profiles of these viruses. WEHV1 and WEHV2 are large, complex retroviruses of 12,999 and 13,125 kb in length, respectively, that are closely related to one another and to walleye dermal sarcoma virus (WDSV). These walleye retroviruses contain three open reading frames, orfA, orfB, and orfC, in addition to gag, pol, and env. orfA and orfB are adjacent to one another and located downstream of env. The OrfA proteins were previously identified as cyclin D homologs that may contribute to the induction of cell proliferation leading to epidermal hyperplasia and dermal sarcoma. The sequence analysis of WEHV1 and WEHV2 revealed that the OrfB proteins are distantly related to the OrfA proteins, suggesting that orfB arose by gene duplication. Presuming that the precursor of orfA and orfB was derived from a cellular cyclin, these genes are the first accessory genes of complex retroviruses that can be traced to a cellular origin. WEHV1, WEHV2, and WDSV are the only retroviruses that have an open reading frame, orfC, of considerable size (ca. 130 amino acids) in the leader region preceding gag. While we were unable to predict a function for the OrfC proteins, they are more conserved than OrfA and OrfB, suggesting that they may be biologically important to the viruses. The transcriptional profiles of WEHV1 and WEHV2 were also similar to that of WDSV; Northern blot analyses detected only low levels of the orfA transcripts in developing lesions, whereas abundant levels of genomic, env, orfA, and orfB transcripts were detected in regressing lesions. The splice donors and acceptors of individual transcripts were identified by reverse transcriptase PCR. The similarities of WEHV1, WEHV2, and WDSV suggest that these viruses use

  2. The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

    PubMed

    Durantel, D; Croizier, L; Ayres, M D; Croizier, G; Possee, R D; López-Ferber, M

    1998-03-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

  3. Kaposi's Sarcoma Associated Herpesvirus Tegument Protein ORF75 Is Essential for Viral Lytic Replication and Plays a Critical Role in the Antagonization of ND10-Instituted Intrinsic Immunity

    PubMed Central

    Full, Florian; Jungnickl, Doris; Reuter, Nina; Bogner, Elke; Brulois, Kevin; Scholz, Brigitte; Stürzl, Michael; Myoung, Jinjong; Jung, Jae U.; Stamminger, Thomas; Ensser, Armin

    2014-01-01

    Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also

  4. The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease.

    PubMed

    Sá Antunes, Tathiana Ferreira; Amaral, Raquel J Vionette; Ventura, José Aires; Godinho, Marcio Tadeu; Amaral, Josiane G; Souza, Flávia O; Zerbini, Poliane Alfenas; Zerbini, Francisco Murilo; Fernandes, Patricia Machado Bueno

    2016-01-01

    Papaya sticky disease, or "meleira", is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2. PMID:27166626

  5. The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease

    PubMed Central

    Sá Antunes, Tathiana Ferreira; Amaral, Raquel J. Vionette; Ventura, José Aires; Godinho, Marcio Tadeu; Amaral, Josiane G.; Souza, Flávia O.; Zerbini, Poliane Alfenas; Zerbini, Francisco Murilo

    2016-01-01

    Papaya sticky disease, or “meleira”, is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2. PMID:27166626

  6. The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease.

    PubMed

    Sá Antunes, Tathiana Ferreira; Amaral, Raquel J Vionette; Ventura, José Aires; Godinho, Marcio Tadeu; Amaral, Josiane G; Souza, Flávia O; Zerbini, Poliane Alfenas; Zerbini, Francisco Murilo; Fernandes, Patricia Machado Bueno

    2016-01-01

    Papaya sticky disease, or "meleira", is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2.

  7. Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

    PubMed

    Yamamoto, Takenobu; Ali, Mir A; Liu, XueQiao; Cohen, Jeffrey I

    2014-03-01

    Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.

  8. Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

    SciTech Connect

    Valles, Steven M.; Hashimoto, Yoshifumi

    2009-06-05

    We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number (FJ528584)), comprised of 10,386 nucleotides, and polyadenylated at the 3' terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5' proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3' proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 +- 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.

  9. Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta.

    PubMed

    Valles, Steven M; Hashimoto, Yoshifumi

    2009-06-01

    We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584), comprised of 10,386 nucleotides, and polyadenylated at the 3' terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5' proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3' proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3+/-1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.

  10. [Serologic studies of domestic cats for potential human pathogenic virus infections from wild rodents].

    PubMed

    Nowotny, N

    1996-05-01

    For several viral infections a reservoir in wild rodents has been demonstrated. Some of the agents are known or suspected to be pathogenic for humans. Because improvements in hygiene have reduced direct human contact with rodents, domestic cats could be acting as active transmitters of these viruses from rodents to man. We selected 4 such pathogens--ortho- and parapox-, hanta- and encephalomyocarditis viruses--which, in different ways, may lead to serious human illness: Ortho- and parapoxvirus infections may cause localized pox lesions following direct skin contact. In general, the lesions heal without complications; in immunosuppressed or -deficient individuals, however, infection may generalize and take a dramatic course. Hantaviruses exist in various serotypes with different pathogenicity for human beings, varying from asymptomatic infection to highly fatal disease. In central and northern Europe the Puumala serotype is predominant causing influenza-like symptoms and renal dysfunction. Human infections arise from inhalation of aerosolized excreta of persistently infected rodents. Infections of man associated with encephalomyocarditis virus were demonstrated sporadically in cases of encephalitis and meningitis. In the present study, we investigated in 200 feline serum samples the prevalence of antibodies to ortho- and parapox-, hanta- and encephalomyocarditis virus. All serum samples were from cats that had been allowed to roam outside and to hunt. They were submitted from all parts of Austria for routine diagnosis in 1993. Four per cent of cats showed antibodies to orthopoxviruses with haemagglutination inhibition (HI) titres of 16-512; because of extensive cross-reactivity, positive samples reacted with all investigated orthopoxviruses (a feline orthopoxvirus recently isolated in Vienna, the reference strain of cowpox virus, Brighton, and vaccinia virus, strain IHD), only varying in titre. The specificity of the results was confirmed by virus neutralisation (VN

  11. Complete nucleotide sequence of Nootka lupine vein-clearing virus.

    PubMed

    Robertson, Nancy L; Côté, Fabien; Paré, Christine; Leblanc, Eric; Bergeron, Michel G; Leclerc, Denis

    2007-12-01

    The complete genome sequence of Nootka lupine vein-clearing virus (NLVCV) was determined to be 4,172 nucleotides in length containing four open reading frames (ORFs) with a similar genetic organization of virus species in the genus Carmovirus, family Tombusviridae. The order and gene product size, starting from the 5'-proximal ORF consisted of: (1) polymerase/replicase gene, ORF1 (p27) and ORF1RT (readthrough) (p87), (2) movement proteins ORF2 (p7) and ORF3 (p9), and, (3) the 3'-proximal coat protein ORF4, (p37). The genomic 5'- and 3'-proximal termini contained a short (59 nt) and a relatively longer 405 nt untranslated region, respectively. The longer replicase gene product contained the GDD motif common to RNA-dependent RNA polymerases. Phylogenetically, NLVCV formed a subgroup with the following four carmoviruses when separately comparing the amino acids of the coat protein or replicase protein: Angelonia flower break virus (AnFBV), Carnation mottle virus (CarMV), Pelargonium flower break virus (PFBV), and Saguaro cactus virus (SgCV). Whole genome nucleotide analysis (percent identities) among the carmoviruses with NLVCV suggested a similar pattern. The species demarcation criteria in the genus Carmovirus for the amino acid sequence identity of the polymerase (<52%) and coat (<41%) protein genes restricted NLVCV as a distinct species, and instead, placed it as a tentative strain of CarMV, PFBV, or SgCV when both the polymerase and CP were used as the determining factors. In contrast, the species criteria that included different host ranges with no overlap and lack of serology relatedness between NLVCV and the carmoviruses, suggested that NLVCV was a distinct species. The relatively low cutoff percentages allowed for the polymerase and CP genes to dictate the inclusion/exclusion of a distinct carmovirus species should be reevaluated. Therefore, at this time we have concluded that NLVCV should be classified as a tentative new species in the genus Carmovirus

  12. [Differentiation of geographic biovariants of smallpox virus by PCR].

    PubMed

    Babkin, I V; Babkina, I N

    2010-01-01

    Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

  13. Polymerization and nucleic acid-binding properties of human L1 ORF1 protein.

    PubMed

    Callahan, Kathryn E; Hickman, Alison B; Jones, Charles E; Ghirlando, Rodolfo; Furano, Anthony V

    2012-01-01

    The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference-the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival. PMID:21937507

  14. A Pilot Study for Standardizing Curriculum-Based Measurement Oral Reading Fluency (CBM ORF) in Arabic

    ERIC Educational Resources Information Center

    Abu-Hamour, Bashir

    2014-01-01

    This study examined the psychometric proprieties of the Arabic version of the Curriculum-Based Measurement Oral Reading Fluency (CBM ORF) for Jordanian students. A sample of 200 students (six to eight years old) was recruited from four public primary schools in Jordan. Results indicated that the CBM ORF had adequate reliability and validity…

  15. Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish

    PubMed Central

    Chew, Guo-Liang; Pauli, Andrea; Schier, Alexander F.

    2016-01-01

    Upstream open reading frames (uORFs) are ubiquitous repressive genetic elements in vertebrate mRNAs. While much is known about the regulation of individual genes by their uORFs, the range of uORF-mediated translational repression in vertebrate genomes is largely unexplored. Moreover, it is unclear whether the repressive effects of uORFs are conserved across species. To address these questions, we analyse transcript sequences and ribosome profiling data from human, mouse and zebrafish. We find that uORFs are depleted near coding sequences (CDSes) and have initiation contexts that diminish their translation. Linear modelling reveals that sequence features at both uORFs and CDSes modulate the translation of CDSes. Moreover, the ratio of translation over 5′ leaders and CDSes is conserved between human and mouse, and correlates with the number of uORFs. These observations suggest that the prevalence of vertebrate uORFs may be explained by their conserved role in repressing CDS translation. PMID:27216465

  16. Erythema Multiforme as a Result of Orf Disease; a Case Report

    PubMed Central

    Biazar, Tahmine; shokri, Mehran; Hosseinnia, Hajar; Bayani, Masomeh

    2016-01-01

    Orf is a mucocutaneous disease that occurs when non-intact skin comes into contact with contaminated sheep saliva. The lesions may complicate to lymphangitis or secondary bacterial infection, but systemic complications such as erythema multiforme, maculopapular rash, and generalized lymphadenopathy are rare. In this paper, we present two cases of erythema multiforme following Orf disease. PMID:27299148

  17. Expression, purification and antibody preparation of PCV2 Rep and ORF3 proteins.

    PubMed

    Peng, Zhiyuan; Ma, Teng; Pang, Daxin; Su, Dan; Chen, Fuwang; Chen, Xinrong; Guo, Ning; Ouyang, Ting; Ouyang, Hongsheng; Ren, Linzhu

    2016-05-01

    Rep and ORF3 proteins are important functional proteins of porcine circovirus 2 (PCV2). Here, Rep and ORF3 genes were cloned, expressed and used to raise polyclonal antibodies. The result showed the recombinant plasmids of Rep and ORF3 genes constructed in this study were expressed efficiently in the prokaryotic system, and the recombinant proteins had antigenicity and immunogenicity. Furthermore, reactivity and specificity of the antiserums were characterized by western blot and indirect immunofluorescent assays. The results elucidated that polyclonal antiserum prepared with Rep or ORF3 had good reactivity and specificity against PCV2, or the Rep and ORF3 expressed in PK-15 cells, respectively. The Rep protein is promising for PCV2 antibody and vaccine development. These results will be helpful for further studies focusing on pathogenesis of PCV2 and serology diagnostic test or vaccine development against PCV2. PMID:26812108

  18. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    PubMed

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (<5%) of ORF57 RNA undergoes double splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein.

  19. Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification.

    PubMed

    Alekseyenko, Artyom A; Gorchakov, Andrey A; Kharchenko, Peter V; Kuroda, Mitzi I

    2014-02-18

    Understanding the composition of epigenetic regulators remains an important challenge in chromatin biology. Traditional biochemical analysis of chromatin-associated complexes requires their release from DNA under conditions that can also disrupt key interactions. Here we develop a complementary approach (BioTAP-XL), in which cross-linking (XL) enhances the preservation of protein interactions and also allows the analysis of DNA targets under the same tandem affinity purification (BioTAP) regimen. We demonstrate the power of BioTAP-XL through analysis of human EZH2, a core subunit of polycomb repressive complex 2 (PRC2). We identify and validate two strong interactors, C10orf12 and C17orf96, which display enrichment with EZH2-BioTAP at levels similar to canonical PRC2 components (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each binding pattern with the location of EZH2 and the H3K27me3-silencing mark, validating their physical interaction with PRC2 components. Interestingly, analysis by mass spectrometry of C10orf12 and C17orf96 interactions revealed that these proteins may be mutually exclusive PRC2 subunits that fail to interact with each other or with JARID2 and AEBP2. C10orf12, in addition, shows a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another histone methyltransferase. Similarly, results from CBX4-BioTAP protein pulldowns are consistent with reports of a diversity of PRC1 complexes. Our results highlight the importance of reciprocal analyses of multiple subunits and suggest that iterative use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms.

  20. Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

    PubMed Central

    Wu, Frederick Y.; Ahn, Jin-Hyun; Alcendor, Donald J.; Jang, Won-Jong; Xiao, Jinsong; Hayward, S. Diane; Hayward, Gary S.

    2001-01-01

    Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis. Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase-helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primase-associated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA. These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8 (ORF-K8) protein, which is

  1. Fail-safe mechanism of GCN4 translational control--uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression.

    PubMed

    Gunišová, Stanislava; Valášek, Leoš Shivaya

    2014-05-01

    One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5' reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively.

  2. Association of the Plasma and Tissue Riboflavin Levels with C20orf54 Expression in Cervical Lesions and Its Relationship to HPV16 Infection

    PubMed Central

    Kelimu, Alimujiang; Guo, Xia; Mamtimin, Batur; Abudula, Abuliz; Upur, Halmurat

    2013-01-01

    Riboflavin deficiency can cause a variety of metabolic problems that lead to skin and mucosal disorders. Limited evidence suggests that high intake of riboflavin may reduce overall risks of cancer. However, association of this deficiency with cervical cancer and precancerous lesions are still not definitively known. In this study, we characterized the relationship between plasma and tissue riboflavin levels and C20orf54 protein expression in patients with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) as well as the relationship of these levels with human papillomavirus virus 16, 18 (HPV16/18) infections. High-performance liquid chromatography (HPLC) was used to measure blood riboflavin levels in patients with CIN and CSCC, and an enzyme-linked immunosorbent assay (ELISA) was used to determine tissue riboflavin levels in patients with CSCC and matched normal mucous epithelia. The expression of C20orf54 in fresh CSCC and matched tissues were detected by qRT-PCR and western blot, respectively. And it was further confirmed by immunohistochemistry (IHC) with formalin-fixed, paraffin-embedded CIN and CSCC. An HPV genotyping chip was used to analyze HPV infection and typing. The results showed that patients with CIN and CSCC had decreased plasma riboflavin levels as compared with normal controls. There was also significantly decreased riboflavin in tissues from CSCC patients, when compared with normal cervical epithelia. C20orf54 expression were significantly up-regulated in CSCC compared to matched control on both mRNA and protein level. Tissue riboflavin levels were significantly lower in HPV16/18 positive tissue compared with HPV16/18-negative tissue, and an inverse association was found between tissue riboflavin levels and C20orf54 mRNA and protein expression in CSCC. Additionally, C20orf54 was significantly correlated with tumor stages. In conclusion, C20orf54 tend to play a protective role in Uyghur cervical carcinogenesis of

  3. The Apis mellifera Filamentous Virus Genome

    PubMed Central

    Gauthier, Laurent; Cornman, Scott; Hartmann, Ulrike; Cousserans, François; Evans, Jay D.; de Miranda, Joachim R.; Neumann, Peter

    2015-01-01

    A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family. PMID:26184284

  4. The Apis mellifera Filamentous Virus Genome.

    PubMed

    Gauthier, Laurent; Cornman, Scott; Hartmann, Ulrike; Cousserans, François; Evans, Jay D; de Miranda, Joachim R; Neumann, Peter

    2015-07-01

    A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

  5. The Apis mellifera filamentous virus genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double strand DNA molecule of approximately 498’500 nucleotides with a GC content of 50.8%. It encompasses 251 non overlapping open reading frames (ORFs), e...

  6. Altered body schema processing in frontotemporal dementia with C9ORF72 mutations

    PubMed Central

    Downey, Laura E; Fletcher, Phillip D; Golden, Hannah L; Mahoney, Colin J; Agustus, Jennifer L; Schott, Jonathan M; Rohrer, Jonathan D; Beck, Jonathan; Mead, Simon; Rossor, Martin N; Crutch, Sebastian J; Warren, Jason D

    2014-01-01

    Background Mutations in C9ORF72 are an important cause of frontotemporal dementia (FTD) and motor neuron disease. Accumulating evidence suggests that FTD associated with C9ORF72 mutations (C9ORF72-FTD) is distinguished clinically by early prominent neuropsychiatric features that might collectively reflect deranged body schema processing. However, the pathophysiology of C9ORF72-FTD has not been elucidated. Methods We undertook a detailed neurophysiological investigation of five patients with C9ORF72-FTD, in relation to patients with FTD occurring sporadically and on the basis of mutations in the microtubule-associated protein tau gene and healthy older individuals. We designed or adapted behavioural tasks systematically to assess aspects of somatosensory body schema processing (tactile discrimination, proprioceptive and body part illusions and self/non-self differentiation). Results Patients with C9ORF72-FTD selectively exhibited deficits at these levels of body schema processing in relation to healthy individuals and other patients with FTD. Conclusions Altered body schema processing is a novel, generic pathophysiological mechanism that may link the distributed cortico-subcortical network previously implicated in C9ORF72-FTD with a wide range of neuropsychiatric and behavioural symptoms, and constitute a physiological marker of this neurodegenerative proteinopathy. PMID:24521566

  7. Axial Spondylometaphyseal Dysplasia Is Caused by C21orf2 Mutations

    PubMed Central

    Nishiguchi, Koji M.; Fujita, Kosuke; Nakazawa, Toru; Alswaid, Abdulrahman; Albalwi, Mohammed A.; Kim, Ok-Hwa; Cho, Tae-Joon; Lim, Gye-Yeon; Isidor, Bertrand; David, Albert; Rustad, Cecilie F.; Merckoll, Else; Westvik, Jostein; Stattin, Eva-Lena; Grigelioniene, Giedre; Kou, Ikuyo; Nakajima, Masahiro; Ohashi, Hirohumi; Smithson, Sarah; Matsumoto, Naomichi; Nishimura, Gen; Ikegawa, Shiro

    2016-01-01

    Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina. PMID:26974433

  8. OrfM: a fast open reading frame predictor for metagenomic data

    PubMed Central

    Woodcroft, Ben J.; Boyd, Joel A.

    2016-01-01

    Summary: Finding and translating stretches of DNA lacking stop codons is a task common in the analysis of sequence data. However, the computational tools for finding open reading frames are sufficiently slow that they are becoming a bottleneck as the volume of sequence data grows. This computational bottleneck is especially problematic in metagenomics when searching unassembled reads, or screening assembled contigs for genes of interest. Here, we present OrfM, a tool to rapidly identify open reading frames (ORFs) in sequence data by applying the Aho–Corasick algorithm to find regions uninterrupted by stop codons. Benchmarking revealed that OrfM finds identical ORFs to similar tools (‘GetOrf’ and ‘Translate’) but is four-five times faster. While OrfM is sequencing platform-agnostic, it is best suited to large, high quality datasets such as those produced by Illumina sequencers. Availability and Implementation: Source code and binaries are freely available for download at http://github.com/wwood/OrfM or through GNU Guix under the LGPL 3+ license. OrfM is implemented in C and supported on GNU/Linux and OSX. Contacts: b.woodcroft@uq.edu.au Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153669

  9. Axial Spondylometaphyseal Dysplasia Is Caused by C21orf2 Mutations.

    PubMed

    Wang, Zheng; Iida, Aritoshi; Miyake, Noriko; Nishiguchi, Koji M; Fujita, Kosuke; Nakazawa, Toru; Alswaid, Abdulrahman; Albalwi, Mohammed A; Kim, Ok-Hwa; Cho, Tae-Joon; Lim, Gye-Yeon; Isidor, Bertrand; David, Albert; Rustad, Cecilie F; Merckoll, Else; Westvik, Jostein; Stattin, Eva-Lena; Grigelioniene, Giedre; Kou, Ikuyo; Nakajima, Masahiro; Ohashi, Hirohumi; Smithson, Sarah; Matsumoto, Naomichi; Nishimura, Gen; Ikegawa, Shiro

    2016-01-01

    Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina. PMID:26974433

  10. Axial Spondylometaphyseal Dysplasia Is Caused by C21orf2 Mutations.

    PubMed

    Wang, Zheng; Iida, Aritoshi; Miyake, Noriko; Nishiguchi, Koji M; Fujita, Kosuke; Nakazawa, Toru; Alswaid, Abdulrahman; Albalwi, Mohammed A; Kim, Ok-Hwa; Cho, Tae-Joon; Lim, Gye-Yeon; Isidor, Bertrand; David, Albert; Rustad, Cecilie F; Merckoll, Else; Westvik, Jostein; Stattin, Eva-Lena; Grigelioniene, Giedre; Kou, Ikuyo; Nakajima, Masahiro; Ohashi, Hirohumi; Smithson, Sarah; Matsumoto, Naomichi; Nishimura, Gen; Ikegawa, Shiro

    2016-01-01

    Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina.

  11. Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

    PubMed

    Torruella, M; Gordon, K; Hohn, T

    1989-10-01

    Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed. PMID:2684630

  12. Characterization of Coffee ringspot virus-Lavras: A model for an emerging threat to coffee production and quality

    SciTech Connect

    Ramalho, T.O.; Figueira, A.R.; Sotero, A.J.; Wang, R.; Geraldino Duarte, P.S.; Farman, M.; Goodin, M.M.

    2014-09-15

    The emergence of viruses in Coffee (Coffea arabica and Coffea canephora), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552 nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945 nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a member of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified. - Highlights: • We report genome sequence determination for Coffee ringspot virus (CoRSV). • CoRSV should be considered a member of the proposed Dichorhavirus genus. • We report temperature-dependent systemic infection of an initially local lesion host. • We report in planta protein and localization data for five CoRSV proteins. • In silico predictions of the CoRSV proteins were validated using in vivo assays.

  13. C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

    PubMed Central

    Muthana, Munitta; Hawtree, Sarah; Wilshaw, Adam; Linehan, Eimear; Roberts, Hannah; Khetan, Sachin; Adeleke, Gbadebo; Wright, Fiona; Akil, Mohammed; Fearon, Ursula; Veale, Douglas; Ciani, Barbara; Wilson, Anthony G.

    2015-01-01

    The variant rs26232, in the first intron of the chromosome 5 open reading frame 30 (C5orf30) locus, has recently been associated with both risk of developing rheumatoid arthritis (RA) and severity of tissue damage. The biological activities of human C5orf30 are unknown, and neither the gene nor protein show significant homology to any other characterized human sequences. The C5orf30 gene is present only in vertebrate genomes with a high degree of conservation, implying a central function in these organisms. Here, we report that C5orf30 is highly expressed in the synovium of RA patients compared with control synovial tissue, and that it is predominately expressed by synovial fibroblast (RASF) and macrophages in the lining and sublining layer of the tissue. These cells play a central role in the initiation and perpetuation of RA and are implicated in cartilage destruction. RASFs lacking C5orf30 exhibit increased cell migration and invasion in vitro, and gene profiling following C5orf30 inhibition confirmed up-regulation of genes involved in cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis model markedly accentuated joint inflammation and tissue damage. Our study reveal C5orf30 to be a previously unidentified negative regulator of tissue damage in RA, and this protein may act by modulating the autoaggressive phenotype that is characteristic of RASFs. PMID:26316022

  14. A novel biomarker C6orf106 promotes the malignant progression of breast cancer.

    PubMed

    Jiang, Guiyang; Zhang, Xiupeng; Zhang, Yong; Wang, Liang; Fan, Chuifeng; Xu, Hongtao; Miao, Yuan; Wang, Enhua

    2015-09-01

    C6orf106 (chromosome 6 open reading frame 106) is a recently discovered protein encoded by the 6th chromosome. Though many proteins encoded by chromosome 6 are reportedly related to cancer, schizophrenia, autoimmunity and many other diseases, the function of C6orf106 was not well demonstrated so far. As measured by immunohistochemical staining, C6orf106 was positive in normal breast duct myoepithelial cells (92.31 %, 72/78), but negative in normal breast duct glandular epithelial cells (3.85 %, 3/78). In breast ductal carcinoma in situ, C6orf106 showed weakly or moderately positive (77.97 %, 46/59), but it was significantly strongly positive in invasive ductal carcinoma (79.57 %, 148/186). The expression intensity of C6orf106 seemed increased significantly along with the malignancy of breast cancer (p < 0.001). Additionally, C6orf106 expression was significantly correlated with TNM stage (p = 0.001 and p = 0.004) and lymph node metastasis (p = 0.018 and p = 0.025) of the overall and the triple-negative breast cancer, respectively. Consistently, we found that the interference of C6orf106 was able to inhibit cell proliferation and invasion of two triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, accompanied by the decrease of cyclin A2, cyclin B1, c-myc, and N-cadherin and the increase of E-cadherin. Collectively, these results indicate that C6orf106 may promote tumor progression in the invasive breast cancer, particularly in triple-negative breast cancer, and C6orf106 might serve as a novel therapeutic target of breast cancer, especially for triple-negative breast cancer.

  15. C9orf72 repeat expansions that cause frontotemporal dementia are detectable among patients with psychosis.

    PubMed

    Watson, Annie; Pribadi, Mochtar; Chowdari, Kodavali; Clifton, Sue; Joel Wood; Miller, Bruce L; Coppola, Giovanni; Nimgaonkar, Vishwajit

    2016-01-30

    A pathologic hexanucleotide repeat expansion in C9orf72 causes frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). Behavioral abnormalities can also occur among mutation carriers with FTD, but it is uncertain whether such mutations occur among persons with psychoses per se. Among participants in a genetic study of psychoses (N=739), two pairs of related individuals had C9orf72 expansions, of whom three were diagnosed with schizophrenia (SZ) / schizoaffective disorder (SZA), but their clinical features did not suggest dementia or ALS. A few patients with SZ/SZA carry C9orf72 repeat expansions; such individuals are highly likely to develop FTD/ALS.

  16. Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations

    PubMed Central

    Rollinson, Sara; Thompson, Jennifer C.; Harris, Jennifer M.; Stopford, Cheryl L.; Richardson, Anna M. T.; Jones, Matthew; Gerhard, Alex; Davidson, Yvonne S.; Robinson, Andrew; Gibbons, Linda; Hu, Quan; DuPlessis, Daniel; Neary, David; Pickering-Brown, Stuart M.

    2012-01-01

    The identification of a hexanucleotide repeat expansion in the C9ORF72 gene as the cause of chromosome 9-linked frontotemporal dementia and motor neuron disease offers the opportunity for greater understanding of the relationship between these disorders and other clinical forms of frontotemporal lobar degeneration. In this study, we screened a cohort of 398 patients with frontotemporal dementia, progressive non-fluent aphasia, semantic dementia or mixture of these syndromes for mutations in the C9ORF72 gene. Motor neuron disease was present in 55 patients (14%). We identified 32 patients with C9ORF72 mutations, representing 8% of the cohort. The patients’ clinical phenotype at presentation varied: nine patients had frontotemporal dementia with motor neuron disease, 19 had frontotemporal dementia alone, one had mixed semantic dementia with frontal features and three had progressive non-fluent aphasia. There was, as expected, a significant association between C9ORF72 mutations and presence of motor neuron disease. Nevertheless, 46 patients, including 22 familial, had motor neuron disease but no mutation in C9ORF72. Thirty-eight per cent of the patients with C9ORF72 mutations presented with psychosis, with a further 28% exhibiting paranoid, deluded or irrational thinking, whereas <4% of non-mutation bearers presented similarly. The presence of psychosis dramatically increased the odds that patients carried the mutation. Mutation bearers showed a low incidence of motor stereotypies, and relatively high incidence of complex repetitive behaviours, largely linked to patients’ delusions. They also showed a lower incidence of acquired sweet food preference than patients without C9ORF72 mutations. Post-mortem pathology in five patients revealed transactive response DNA-binding protein 43 pathology, type A in one patient and type B in three. However, one patient had corticobasal degeneration pathology. The findings indicate that C9ORF72 mutations cause some but not all

  17. Molecular epidemiology of orf513-bearing class 1 integrons in multiresistant clinical isolates from Argentinean hospitals.

    PubMed

    Arduino, Sonia M; Catalano, Mariana; Orman, Betina E; Roy, Paul H; Centrón, Daniela

    2003-12-01

    The spread of orf513-bearing class 1 integrons is associated with bla(CTX-M-2) in gram-negative clinical isolates in Argentina, with In35 being the most frequently found integron (74%). Among 65 isolates without bla(CTX-M-2), only one harbored a novel orf513-bearing class 1 integron with the dfrA3b gene. The finding of orf513 not associated with class 1 integrons in two gram-positive strains indicates the widespread occurrence of this putative site-specific recombinase.

  18. Regulation of the Abundance of Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2

    PubMed Central

    Chang, Tzu-Hsuan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-01-01

    The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. PMID:27698494

  19. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  20. Characterization, nucleotide sequence and genome organization of leek white stripe virus, a putative new species of the genus Necrovirus.

    PubMed

    Lot, H; Rubino, L; Delecolle, B; Jacquemond, M; Turturo, C; Russo, M

    1996-01-01

    White stripe is a disease affecting leek in France with which an isometric virus c. 30 nm in diameter is associated. The most evident symptom is the presence of white stripes on the leaves extending to the stem. Attempts to demonstrate transmission through the soil by sowing or transplanting leek in contaminated soil were unsuccessful. The virus was transmitted by sap inoculation to a narrow range of herbaceous hosts, all of which were infected only locally. Virus purification was from infected leek tissues, where it accumulated in large amounts, as demonstrated by ultrastructural observations. RNA was extracted from purified virus preparations and cDNA clones were prepared. The complete nucleotide sequence of the viral RNA was determined: The genome is 3,662 nucleotides long and contains five open reading frames (ORFs). The first (ORF 1) encodes a putative translation product of M(r) 23,803 (p24) and read through of its amber stop codon results in a protein of M(r) 82,625 (p83) (ORF 2). ORF 3 and ORF 4 encode two small polypeptides of M(r) 11,280 (p11) and M(r) 6,261 (p6), respectively. ORF 5 encodes the capsid protein of M(r) 27,460 (p27). The genome organization and sequence alignments with the corresponding products of necroviruses suggest that the virus isolated from leek is a new species in the genus Necrovirus, for which the name of leek white stripe virus (LWSV) is proposed.

  1. Pri sORF peptides induce selective proteasome-mediated protein processing.

    PubMed

    Zanet, J; Benrabah, E; Li, T; Pélissier-Monier, A; Chanut-Delalande, H; Ronsin, B; Bellen, H J; Payre, F; Plaza, S

    2015-09-18

    A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

  2. Genome of horsepox virus.

    PubMed

    Tulman, E R; Delhon, G; Afonso, C L; Lu, Z; Zsak, L; Sandybaev, N T; Kerembekova, U Z; Zaitsev, V L; Kutish, G F; Rock, D L

    2006-09-01

    Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses.

  3. Roles for lambda Orf and Escherichia coli RecO, RecR and RecF in lambda recombination.

    PubMed

    Sawitzke, J A; Stahl, F W

    1997-10-01

    Bacteriophage lambda lacking its Red recombination functions requires either its own gene product, Orf, or the product of Escherichia coli's recO, recR and recF genes (RecORF) for efficient recombination in recBC sbcB sbcC mutant cells (the RecF pathway). Phage crosses under conditions of a partial block to DNA replication have revealed the following: (1) In the presence of Orf, RecF pathway recombination is similar to lambda Red recombination; (2) Orf is necessary for focusing recombination toward the right end of the chromosome as lambda is conventionally drawn; (3) RecORF-mediated RecF pathway recombination is not focused toward the right end of the chromosome, which may indicate that RecORF travels along the DNA; (4) both Orf- and RecORF-mediated RecF pathway recombination are stimulated by DNA replication; and (5) low level recombination in the simultaneous absence of Orf and RecORF may occur by a break-copy mechanism that is not initiated by a double strand break. Models for the roles of Orf and RecO, RecR and RecF in recombination are presented. PMID:9335578

  4. Molecular characterization of Hop mosaic virus: its serological and molecular relationships to Hop latent virus.

    PubMed

    Hataya, T; Arimoto, R; Suda, N; Uyeda, I

    2001-10-01

    The 3'-terminal sequence of hop mosaic virus (HpMV) genomic RNA was determined. A cDNA of approximately 1.8 kbp was amplified from the HpMV genome by 3' RACE using a degenerate primer, which was designed to anneal to the overlapping region of open reading frames (ORFs) 2 and 3 of eight carlavirus genomes. The sequence contained three ORFs, encoding proteins of 7-, 34-, and 11-kDa, which corresponded to ORFs 4, 5, and 6 of the carlavirus genome, respectively. The amino acid sequence of ORF 5, encoding the coat protein (CP) of HpMV, shows the highest identity (67%) to that of Hop latent virus (HpLV). The HpMV CP N-terminal sequence differs from that of HpLV, but the central and C-terminal sequences of the CP of both viruses are similar. The sequence similarity possibly causes the cross-reaction of heterologous antibodies of HpMV and HpLV. Phylogenetic analyses based on the CP amino acid and 3' non-coding region sequences indicate close relationships among HpMV, HpLV, and Potato virus M. We report here the first molecular characterization of HpMV genomic RNA. PMID:11722015

  5. Repeat expansions in the C9ORF72 gene contribute to Alzheimer's disease in Caucasians.

    PubMed

    Kohli, Martin A; John-Williams, Krista; Rajbhandary, Ruchita; Naj, Adam; Whitehead, Patrice; Hamilton, Kara; Carney, Regina M; Wright, Clinton; Crocco, Elizabeth; Gwirtzman, Harry E; Lang, Rosalyn; Beecham, Gary; Martin, Eden R; Gilbert, John; Benatar, Michael; Small, Gary W; Mash, Deborah; Byrd, Goldie; Haines, Jonathan L; Pericak-Vance, Margaret A; Züchner, Stephan

    2013-05-01

    Recently, a hexanucleotide repeat expansion in the C9ORF72 gene has been identified to account for a significant portion of Caucasian families affected by frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Given the clinical overlap of FTD with Alzheimer's disease (AD), we hypothesized that C9ORF72 expansions might contribute to AD. In Caucasians, we found C9ORF72 expansions in the pathogenic range of FTD/ALS (>30 repeats) at a proportion of 0.76% in AD cases versus 0 in control subjects (p = 3.3E-03; 1182 cases, 1039 controls). In contrast, no large expansions were detected in individuals of African American ethnicity (291 cases, 620 controls). However, in the range of normal variation of C9ORF72 expansions (0-23 repeat copies), we detected significant differences in distribution and mean repeat counts between Caucasians and African Americans. Clinical and pathological re-evaluation of identified C9ORF72 expansion carriers revealed 9 clinical and/or autopsy confirmed AD and 2 FTD final diagnoses. Thus, our results support the notion that large C9ORF72 expansions lead to a phenotypic spectrum of neurodegenerative disease including AD.

  6. Clinicopathological Study of Patients With C9ORF72-Associated Frontotemporal Dementia Presenting With Delusions

    PubMed Central

    Shinagawa, Shunichiro; Naasan, Georges; Karydas, Anna M.; Coppola, Giovanni; Pribadi, Mochtar; Seeley, William W.; Trojanowski, John Q.; Miller, Bruce L.; Grinberg, Lea T.

    2015-01-01

    Background Several clinical studies point to a high prevalence of psychotic symptoms in frontotemporal dementia associated with C9ORF72 mutations, but clinicopathological studies addressing the association between C9ORF72 mutations and delusions are lacking. Method Seventeen patients with pathologically proven frontotemporal lobar degeneration (FTLD) associated with C9ORF72 mutations were identified from Neurodegenerative Disease Brain Bank. Of the 17 cases with C9ORF72 mutation, 4 exhibited well-defined delusions. The clinical history, neurological examination, neuropsychological testing, neuroimaging analysis, and postmortem assessment of the patients with delusions were evaluated and compared with the other cases. Result The content of the delusions was mixed including persecution, infidelity, and grandiosity. All cases showed parkinsonism; voxel-based morphometry analysis showed greater precuneus atrophy in patients with delusions than those without delusions. All 4 had unclassifiable FTLD with TAR DNA-binding protein inclusions, with characteristics of both type A and type B. Three cases had additional τ pathology and another had α-synuclein pathology. Conclusion C9ORF72 carriers with well-defined delusions likely associated with additional pathologies and parietal atrophy in neuroimaging. Patients presenting with middle-aged onset of delusions should be screened for C9ORF72 mutations, especially if family history and parkinsonism are present. PMID:25342578

  7. Display of ISKNV orf086 protein on the surface of Aeromonas hydrophila and its immunogenicity in Chinese perch (Siniperca chuatsi).

    PubMed

    Fu, Xiaozhe; Lin, Qiang; Liu, Lihui; Liang, Hongru; Huang, Zhibin; Li, Ningqiu

    2016-09-01

    Co-infection with infectious spleen and kidney necrosis virus (ISKNV) and Aeromonas hydrophila is becoming ever more widespread in Chinese perch (Siniperca chuatsi) aquaculture industry, so that it's necessary to develop the combined vaccine against ISKNV and A. hydrophila disease. The surface display of heterologous on bacteria using anchoring motifs from outer membranes proteins has already been explored as an effective delivery system of viral antigens. In present study, the ISKNV orf086 gene, which is verified as a protective antigen, was inserted into ompA gene cassette of A. hydrophila GYK1 strain by homologous recombination. And an ompA-orf086 fusion A. hydrophila mutant strain K28 was constructed. Then the ISKNV orf086 was verified to express on the surface of A. hydrophila K28 by RT-PCR, western blot and indirect immunofluorescence assay. Next, Chinese perch were intraperitoneally inoculated with formalin inactivated A. hydrophila k28 emulsified with ISA763 adjuvant with a dose of 9 × 10(8) CFU per fish. Transcriptional analysis of non-specific and specific immune related genes revealed that the expression levels of IRF-7, IRAK1, Mx, Viperin, Lysozyme and IgM were strongly up-regulated in Chinese perch post-inoculation. In addition, specific antibodies were detected by ELISA, and the results showed that antibody titer against ISKNV or A. hydrophila reached the highest with 1:800 or 1:1200 on 14dpv, respectively. Lymphocyte proliferation were detected by MTT methods, and the results showed that the SI values of AH-K28 vaccinated group to three different stimulators were significantly higher than those of control group. At last, protective efficacy were determined by challenge trials. The cumulative mortality rates of vaccinated groups were significantly lower than the control one (P < 0.05) after ISKNV or A. hydrophila challenge, and the relative percentage survival (RPS) value was 73.3% and 60%, respectively. This system provides a novel approach to

  8. Insights into the translational regulation of biologically active open reading frames of Pelargonium line pattern virus.

    PubMed

    Castaño, Aurora; Ruiz, Leticia; Hernández, Carmen

    2009-04-10

    Pelargonium line pattern virus (PLPV), a proposed member of a prospective genus (Pelarspovirus) within family Tombusviridae, has a positive-sense, single-stranded genomic RNA. According to previous predictions, it contains six open reading frames (ORFs) potentially encoding proteins of 27 (p27), 13 (p13), 87 (p87), 7 (p7), 6 (p6), and 37 kDa (p37). Using a variety of techniques we demonstrate that all predicted ORFs are functional, with the exception of (p13) and (p6). We also characterize a previously unidentified ORF which encodes a 9.7 kDa protein (p9.7) that is essential for viral movement. Furthermore, we present evidence that the single subgenomic RNA (sgRNA) produced by the virus directs synthesis of p7, p9.7 and p37. Remarkably, the translation of these totally unrelated proteins is coordinated via leaky-scanning. This mechanism seems to be favoured by the poor translation context of the start codon of ORF(p7), the non-AUG weak initiation codon of ORF(p9.7) and the lack of additional AUG codons in any reading frame preceding ORF(p37). The results also suggest that precise regulation of protein production from the sgRNA is critical for virus viability. Altogether, the data supports the notion that PLPV belongs to a new genus of plant viruses.

  9. Expression of African swine fever virus envelope protein j13L inhibits vaccinia virus morphogenesis.

    PubMed

    Jacobs, S C; Dixon, L K; Brookes, S M; Smith, G L

    1998-05-01

    The African swine fever virus (ASFV) strain Malawi LIL20/1 open reading frame (ORF) j13L was expressed in vaccinia virus (VV) from a strong synthetic late promoter as either a complete ORF (vSJ1) or lacking codons 1-31 (vSJ2). Each recombinant VV produced a small plaque which rapidly reverted to a normal size upon passage. The yield of infectious virus from a single cycle infection with vSJ1 or vSJ2 was reduced 50- to 100-fold compared to wild-type (wt) and a revertant virus (vSJ5) in which the j13L ORF was removed and the VV thymidine kinase gene restored. PCR analysis of nine spontaneous large plaque revertant viruses, recovered after passage of vSJ1 in BSC-40 cells, showed that six had lost the j13L ORF and the co-inserted beta-galactosidase gene. Three viruses retained the j13L and beta-galactosidase genes, but in each case the j13L protein was not expressed due to a different single base deletion near the 5' end of the j13L coding region which introduced a stop codon a short distance downstream. The formation of intracellular mature virus (IMV) and extracellular enveloped virus was reduced 50- to 75-fold in cells infected with vSJ1 compared to wt VV and revertant vSJ5. Electron microscopy showed aberrant IMV precursor structures in vSJ1-infected cells, and immunoelectron microscopy demonstrated that these structures contained j13L protein. These results indicate that expression of the j13L protein is toxic for VV replication due to interference with VV morphogenesis prior to IMV formation.

  10. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for

  11. Low dependency of retrotransposition on the ORF1 protein of the zebrafish LINE, ZfL2-1.

    PubMed

    Kajikawa, Masaki; Sugano, Tomohiro; Sakurai, Ryosuke; Okada, Norihiro

    2012-05-10

    The zebrafish long interspersed element (LINE), ZfL2-1, which belongs to the L2 clade, contains two open reading frames, ORF1 and ORF2. ORF1 encodes a protein containing a coiled-coil motif and an esterase domain, whereas ORF2 encodes a protein containing an endonuclease and a reverse transcriptase domain. To elucidate the functional significance of ORF1 in retrotransposition, we constructed many variants of ZfL2-1 and examined their retrotransposition ability. We concluded: 1) the ORF1 protein is not essential for ZfL2-1 retrotransposition in cultured cells; 2) the translation of ORF1 is required for the translation of ORF2; and 3) ORF2 translation probably occurs via suppression of the ORF1 stop codon, the efficiency of which is influenced by the context of the sequence juxtaposed to the 3' side of the stop codon. These results offer a new perspective on the evolution of the L2 clade LINEs. PMID:22405944

  12. Structure and Mechanism of ORF36, an Amino Sugar Oxidizing Enzyme in Everninomicin Biosynthesis

    SciTech Connect

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T.M.

    2010-12-07

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, L-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-L-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-L-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of {sup 18}O{sub 2} establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 {angstrom} resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

  13. Structure and mechanism of ORF36, an Aminosugar Oxidizing Enzyme in Everninomicin Biosynthesis†

    PubMed Central

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T. M.

    2010-01-01

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitrosugar, l-evernitrose, analogs of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically-generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of 18O2 establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products, and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 Å resolution x-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-coA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36. PMID:20866105

  14. Bromodomain inhibitors regulate the C9ORF72 locus in ALS.

    PubMed

    Zeier, Zane; Esanov, Rustam; Belle, Kinsley C; Volmar, Claude-Henry; Johnstone, Andrea L; Halley, Paul; DeRosa, Brooke A; Khoury, Nathalie; van Blitterswijk, Marka; Rademakers, Rosa; Albert, Jeffrey; Brothers, Shaun P; Wuu, Joanne; Dykxhoorn, Derek M; Benatar, Michael; Wahlestedt, Claes

    2015-09-01

    A hexanucleotide repeat expansion residing within the C9ORF72 gene represents the most common known cause of amyotrophic lateral sclerosis (ALS) and places the disease among a growing family of repeat expansion disorders. The presence of RNA foci, repeat-associated translation products, and sequestration of RNA binding proteins suggests that toxic RNA gain-of-function contributes to pathology while C9ORF72 haploinsufficiency may be an additional pathological factor. One viable therapeutic strategy for treating expansion diseases is the use of small molecule inhibitors of epigenetic modifier proteins to reactivate expanded genetic loci. Indeed, previous studies have established proof of this principle by increasing the drug-induced expression of expanded (and abnormally heterochromatinized) FMR1, FXN and C9ORF72 genes in respective patient cells. While epigenetic modifier proteins are increasingly recognized as druggable targets, there have been few screening strategies to address this avenue of drug discovery in the context of expansion diseases. Here we utilize a semi-high-throughput gene expression based screen to identify siRNAs and small molecule inhibitors of epigenetic modifier proteins that regulate C9ORF72 RNA in patient fibroblasts, lymphocytes and reprogrammed motor neurons. We found that several bromodomain small molecule inhibitors increase the expression of C9ORF72 mRNA and pre-mRNA without affecting repressive epigenetic signatures of expanded C9ORF72 alleles. These data suggest that bromodomain inhibition increases the expression of unexpanded C9ORF72 alleles and may therefore compensate for haploinsufficiency without increasing the production of toxic RNA and protein products, thereby conferring therapeutic value. PMID:26099177

  15. Bromodomain inhibitors regulate the C9ORF72 locus in ALS

    PubMed Central

    Zeier, Zane; Esanov, Rustam; Belle, Kinsley C.; Volmar, Claude-Henry; Johnstone, Andrea L.; Halley, Paul; DeRosa, Brooke A.; Khoury, Nathalie; van Blitterswijk, Marka; Rademakers, Rosa; Albert, Jeffrey; Brothers, Shaun P.; Wuu, Joanne; Dykxhoorn, Derek M.; Benatar, Michael; Wahlestedt, Claes

    2015-01-01

    A hexanucleotide repeat expansion residing within the C9ORF72 gene represents the most common known cause of amyotrophic lateral sclerosis (ALS) and places the disease among a growing family of repeat expansion disorders. The presence of RNA foci, repeat-associated translation products, and sequestration of RNA binding proteins suggests that toxic RNA gain-of-function contributes to pathology while C9ORF72 haploinsufficiency may be an additional pathological factor. One viable therapeutic strategy for treating expansion diseases is the use of small molecule inhibitors of epigenetic modifier proteins to reactivate expanded genetic loci. Indeed, previous studies have established proof of this principle by increasing the drug-induced expression of expanded (and abnormally heterochromatinized) FMR1, FXN and C9ORF72 genes in respective patient cells. While epigenetic modifier proteins are increasingly recognized as druggable targets, there have been few screening strategies to address this avenue of drug discovery in the context of expansion diseases. Here we utilize a semi-high-throughput gene expression based screen to identify siRNAs and small molecule inhibitors of epigenetic modifier proteins that regulate C9ORF72 RNA in patient fibroblasts, lymphocytes and reprogrammed motor neurons. We found that several bromodomain small molecule inhibitors increase the expression of C9ORF72 mRNA and pre-mRNA without affecting repressive epigenetic signatures of expanded C9ORF72 alleles. These data suggest that bromodomain inhibition increases the expression of unexpanded C9ORF72 alleles and may therefore compensate for haploinsufficiency without increasing the production of toxic RNA and protein products, thereby conferring therapeutic value. PMID:26099177

  16. Rewiring the RNAs of influenza virus to prevent reassortment

    PubMed Central

    Gao, Qinshan; Palese, Peter

    2009-01-01

    Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses. PMID:19805230

  17. Each of the eight simian hemorrhagic fever virus minor structural proteins is functionally important

    PubMed Central

    Vatter, Heather A.; Di, Han; Donaldson, Eric F.; Baric, Ralph S.; Brinton, Margo A.

    2014-01-01

    The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant. PMID:25036340

  18. Production and characterization of a Brazilian candidate antigen for Hepatitis E Virus genotype 3 diagnosis.

    PubMed

    de Almeida Ramos, Denise; Miani, Michela; Pandolfi, Rafael; Tondo, Luis; Colli, Maikel L; Rosado Spilki, Fernando; Rovaris Gardinali, Noemi; Alves Pinto, Marcelo; Kreutz, Luiz C; Frandoloso, Rafael

    2016-03-01

    Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2((394-661)) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets' feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p.

  19. Coding capacity determines in vivo accumulation of a defective RNA of clover yellow mosaic virus.

    PubMed Central

    White, K A; Bancroft, J B; Mackie, G A

    1992-01-01

    Naturally occurring defective RNAs (D RNAs) derived from the potexvirus clover yellow mosaic virus (CYMV) contain large internal deletions yet maintain a single open reading frame (ORF) representing the in-frame fusion of 5' and 3' terminal ORFs. Capped transcripts of the prototype 1.2-kb D RNA of CYMV were synthesized in vitro and used to inoculate broad bean plants. Progeny D RNA accumulated only if synthetic D RNA transcripts were coinoculated with CYMV RNA. Several experiments showed that helper-dependent accumulation of the D RNA in vivo depended on the maintenance of its encoded fusion ORF. (i) D RNAs with six-residue deletions introduced early in the fusion ORF accumulated, whereas those with four-residue out-of-frame deletions at the same sites were nonviable. (ii) Analysis of D RNAs containing termination codons at different locations showed that only the most 3' stop codon (maintaining over 93% of the fusion ORF) was permissive for D RNA accumulation. (iii) D RNAs with small in-frame deletions and insertions in their 3' coding regions were viable. (iv) Nonviable D RNAs containing disrupted fusion ORFs could not be complemented by the presence in the infection of a D RNA encoding a complete fusion ORF. Taken together, the results indicate that the process of translation, rather than the encoded product, modulates an event(s) which influences the propagation and/or accumulation of this RNA in vivo. This represents a unique requirement among plant virus D RNAs. Images PMID:1560537

  20. Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

    PubMed Central

    Kiss-László, Z; Blanc, S; Hohn, T

    1995-01-01

    A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV. Images PMID:7628455

  1. New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia.

    PubMed

    Tang, Kathy F J; Navarro, Solangel A; Pantoja, Carlos R; Aranguren, Fernando L; Lightner, Donald V

    2012-07-25

    White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly pathogenic to penaeid shrimp and have caused significant economic losses in the shrimp culture industry around the world. During 2010 and 2011, both WSSV and TSV were found in Saudi Arabia, where they caused severe mortalities in cultured Indian white shrimp Penaeus indicus. Most outbreaks of shrimp viruses in production facilities can be traced to the importation of infected stocks or commodity shrimp. In an attempt to determine the origins of these viral outbreaks in Saudi Arabia, we performed variable number of tandem repeat (VNTR) analyses for WSSV isolates and a phylogenetic analysis for TSV isolates. From the WSSV genome, the VNTR in open reading frames (ORFs) 125 and 94 were investigated with PCR followed by DNA sequence analysis. The genotypes were categorized as {N125, N94} where N is the number of repeat units in a specific ORF, and the subscript indicates the ORF (i.e. ORFs 125 and 94 in this case). From 15 Saudi Arabia WSSV isolates, we detected 3 genotypes: {6125, 794}, {7125, del94}, and {8125, 1394}. The WSSV genotype of {7125, del94} appears to be a new variant with a 1522 bp deletion encompassing complete coding regions of ORF 94 and ORF 95 and the first 82 bp of ORF 93. For TSV genotyping, we used a phylogenetic analysis based on the amino acid sequence of TSV capsid protein 2 (CP2). We analyzed 8 Saudi Arabian isolates in addition to 36 isolates from other areas: SE Asia, Mexico, Venezuela and Belize. The Saudi Arabian TSV clustered into a new, distinct group. Based on these genotyping analyses, new WSSV and TSV genotypes were found in Saudi Arabia. The data suggest that they have come from wild shrimp Penaeus indicus from the Red Sea that are used for broodstock. PMID:22832716

  2. Psychosis and Hallucinations in FTD with C9ORF72 mutation: A detailed clinical cohort

    PubMed Central

    Kertesz, Andrew; Ang, Lee Cyn; Jesso, Sarah; MacKinley, Julia; Baker, Matt; Brown, Patricia; Shoesmith, Christen; Rademakers, Rosa; Finger, Elizabeth C.

    2014-01-01

    OBJECTIVE To describe in detail the presenting symptoms and clinical course of a cohort of patients with Frontotemporal dementia and the recently described C9ORF72 repeat expansion. BACKGROUND Recent discovery of the C9ORF72 repeat expansion linked to familial frontotemporal dementia and ALS has permitted retrospective evaluation of potential defining clinical characteristics that may distinguish C9ORF72 mutation carriers from other patients with FTD. Prior reports have identified a subset of patients with an increased incidence of psychosis, specifically delusions, though the detailed nature of these symptoms is not yet well described. METHODS We conducted a retrospective chart review of to report the detailed case histories of 7 patients with C9ORF72 mutations from a cohort of 61 patients with FTD. Results Detailed histories available from these patients reveal an increased incidence of psychosis, including visual and auditory hallucinations and delusions compared to sporadic FTD patients in our cohort. CONCLUSIONS This cohort confirms and adds symptom-related details to prior reports of increased incidence of psychotic phenomenon in FTD and ALS patients with C9ORF72 mutations, to enhance future clinical identification and diagnosis of patients presenting with these symptoms. PMID:24077574

  3. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

    PubMed

    Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J; Ivison, David; Généreux, Catherine; Dubus, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J; Lloyd, Adrian J; Roper, David I; Schofield, Christopher J; Andersson, Inger; McDonough, Michael A

    2013-08-01

    Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

  4. C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD.

    PubMed

    O'Rourke, Jacqueline G; Bogdanik, Laurent; Muhammad, A K M G; Gendron, Tania F; Kim, Kevin J; Austin, Andrew; Cady, Janet; Liu, Elaine Y; Zarrow, Jonah; Grant, Sharday; Ho, Ritchie; Bell, Shaughn; Carmona, Sharon; Simpkinson, Megan; Lall, Deepti; Wu, Kathryn; Daughrity, Lillian; Dickson, Dennis W; Harms, Matthew B; Petrucelli, Leonard; Lee, Edward B; Lutz, Cathleen M; Baloh, Robert H

    2015-12-01

    Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (∼100-1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients. PMID:26637796

  5. Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans.

    PubMed

    Pueyo, José I; Magny, Emile G; Sampson, Christopher J; Amin, Unum; Evans, Iwan R; Bishop, Sarah A; Couso, Juan P

    2016-03-01

    Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease. PMID:27015288

  6. Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans

    PubMed Central

    Pueyo, José I.; Amin, Unum; Evans, Iwan R.; Bishop, Sarah A.; Couso, Juan P.

    2016-01-01

    Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease. PMID:27015288

  7. Human sealpox resulting from a seal bite: confirmation that sealpox virus is zoonotic.

    PubMed

    Clark, C; McIntyre, P G; Evans, A; McInnes, C J; Lewis-Jones, S

    2005-04-01

    The case of a marine mammal technician who sustained a seal-bite to the hand that produced a lesion clinically very similar to orf is described. Sequence analysis of the viral DNA amplified from the lesion by the polymerase chain reaction indicated that it was sealpox virus in origin. This is the first report providing unequivocal evidence that sealpox may be transmitted to humans and causes lesions very similar to orf. PMID:15840117

  8. A shutoff and exonuclease mutant of murine gammaherpesvirus-68 yields infectious virus and causes RNA loss in type I interferon receptor knockout cells.

    PubMed

    Sheridan, Victoria; Polychronopoulos, Louise; Dutia, Bernadette M; Ebrahimi, Bahram

    2014-05-01

    Significant loss of RNA followed by severely reduced cellular protein pool, a phenomenon termed host shutoff, is associated with a number of lytic virus infections and is a critical player in viral pathogenesis. Until recently, viral DNA exonucleases were associated only with processing of viral genomic DNA and its encapsidation. However, recent observations have identified host shutoff and exonuclease function for the highly conserved viral exonucleases in γ-herpesviruses, which include Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus and the mouse model murine gammaherpesvirus-68, also referred to as MHV-68. In this study, we show that although ablation of the MHV-68 exonuclease ORF37 caused a restrictive phenotype in WT IFN-α/β receptor-positive cells such as NIH 3T3, lack of ORF37 was tolerated in cells lacking the IFN-α/β receptor: the ORF37Stop virus was capable of forming infectious particles and caused loss of mRNA in IFN-α/β receptor knockout cells. Moreover, ORF37Stop virus was able to establish lytic infection in the lungs of mice lacking the IFN-α/β receptor. These observations provide evidence that lytic MHV-68 infection and subsequent loss of mRNA can take place independently of ORF37. Moreover, efficient growth of ORF37Stop virus also identifies a role for this family of viral nucleases in providing a window of opportunity for virus growth by overcoming type I IFN-dependent responses.

  9. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  10. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  11. Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation

    PubMed Central

    De Luca, Chiara; Guadagni, Fiorella; Sinibaldi-Vallebona, Paola; Sentinelli, Steno; Gallucci, Michele; Hoffmann, Andreas; Schumann, Gerald G.; Spadafora, Corrado; Sciamanna, Ilaria

    2016-01-01

    LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer. We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues. We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively. Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker. PMID:26716650

  12. Mutations in c12orf57 cause a syndromic form of colobomatous microphthalmia.

    PubMed

    Zahrani, Fatema; Aldahmesh, Mohammed A; Alshammari, Muneera J; Al-Hazzaa, Selwa A F; Alkuraya, Fowzan S

    2013-03-01

    Microphthalmia is an important developmental eye disorder. Although mutations in several genes have been linked to this condition, they only account for a minority of cases. We performed autozygome analysis and exome sequencing on a multiplex consanguineous family in which colobomatous microphthalmia is associated with profound global developmental delay, intractable seizures, and corpus callosum abnormalities, and we identified a homozygous truncating mutation in C12orf57 [c.1A>G; p.Met1?]. In a simplex case with a similar phenotype, we identified compound heterozygosity for the same mutation and another missense mutation [c.152T>A; p.Leu51Gln]. Little is known about C12orf57 but we show that it is expressed in several mouse tissues, including the eye and brain. Our data strongly implicate mutations in C12orf57 in the pathogenesis of a clinically distinct autosomal-recessive syndromic form of colobomatous microphthalmia.

  13. Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.

    PubMed

    Bazzini, Ariel A; Johnstone, Timothy G; Christiano, Romain; Mackowiak, Sebastian D; Obermayer, Benedikt; Fleming, Elizabeth S; Vejnar, Charles E; Lee, Miler T; Rajewsky, Nikolaus; Walther, Tobias C; Giraldez, Antonio J

    2014-05-01

    Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

  14. Frontotemporal lobar dementia and amyotrophic lateral sclerosis associated with c9orf72 expansion.

    PubMed

    Le Ber, I

    2015-01-01

    An intronic GGGGCC repeat expansion in c9orf72 gene has been identified as the most common genetic cause of frontotemporal lobar dementia (FTLD), amyotrophic lateral sclerosis (ALS) and FTLD-ALS. The discovery of c9orf72 gene has led to important scientific progresses and has considerably changed our clinical practice over the last few years. This paper summarizes the common and less typical phenotypes associated with c9orf72 expansion, the complex pathological pattern characterized by p62/dipeptide repeat aggregates, as well as the pathological mechanisms by which the expansion might produce neurodegeneration implicating loss-of-function, RNA toxicity, RNA-binding protein sequestration and accumulation of dipeptide repeats. We also discuss the recommendations and limits for genetic testing and counseling in clinical practice. PMID:26032484

  15. Rubus chlorotic mottle virus, a new sobemovirus infecting raspberry and bramble.

    PubMed

    McGavin, W J; Macfarlane, S A

    2009-01-01

    The complete nucleotide sequence of a new member of the unassigned genus Sobemovirus, isolated from raspberry and bramble plants in north east Scotland and given the name Rubus chlorotic mottle virus (RuCMV), was obtained. The virus has a single, positive-strand RNA genome of 3,983 nucleotides and, in common with other sobemoviruses, contains four open reading frames (ORFs) encoding, from 5' to 3', the P1 protein that is likely to be a suppressor of RNA silencing, ORF2a that has homology to serine-proteases, ORF2b that is the probable RNA dependent RNA polymerase, and ORF3 that is the coat protein. ORF2b protein is potentially expressed as a fusion with ORF2a protein by a -1 frameshift at the heptanucleotide sequence UUUAAAC. Phylogenetic analyses showed that RuCMV is a distinct virus not closely related to any of the other sequenced sobemoviruses. Based on the obtained sequence a full-length cDNA copy of RuCMV was cloned and in vitro transcripts derived from this clone were shown to be fully infectious.

  16. Rubus chlorotic mottle virus, a new sobemovirus infecting raspberry and bramble.

    PubMed

    McGavin, W J; Macfarlane, S A

    2009-01-01

    The complete nucleotide sequence of a new member of the unassigned genus Sobemovirus, isolated from raspberry and bramble plants in north east Scotland and given the name Rubus chlorotic mottle virus (RuCMV), was obtained. The virus has a single, positive-strand RNA genome of 3,983 nucleotides and, in common with other sobemoviruses, contains four open reading frames (ORFs) encoding, from 5' to 3', the P1 protein that is likely to be a suppressor of RNA silencing, ORF2a that has homology to serine-proteases, ORF2b that is the probable RNA dependent RNA polymerase, and ORF3 that is the coat protein. ORF2b protein is potentially expressed as a fusion with ORF2a protein by a -1 frameshift at the heptanucleotide sequence UUUAAAC. Phylogenetic analyses showed that RuCMV is a distinct virus not closely related to any of the other sequenced sobemoviruses. Based on the obtained sequence a full-length cDNA copy of RuCMV was cloned and in vitro transcripts derived from this clone were shown to be fully infectious. PMID:18929604

  17. Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment.

    PubMed

    Schmitt, J F; Stunnenberg, H G

    1988-06-01

    The complete sequence of the vaccinia virus HindIII I fragment, which is composed of 6,498 base pairs, encodes six complete and two incomplete open reading frames (ORFs). Computer analysis revealed an amino acid sequence homology between ORF I 4 and the large subunit of the ribonucleotide reductase complex. The two small polypeptides derived from ORFs I 2 and I 5, with molecular weights of 8,500 and 8,700, respectively, have a very high hydrophobic amino acid sequence composition. S1 analysis revealed that ORF I 4 is expressed at early stages of infection, ORFs I 1, I 2, I 5, and I 7 are expressed in the late phase of infection, and ORF I 3 is constitutively expressed. Screening a vaccinia virus genomic library revealed a large vaccinia virus insert overlapping the HindIII I and O fragments which contains a previously undetected HindIII P fragment of approximately 300 base pairs. S1 analysis revealed an early (O1) and a late (O2) start site of transcription initiation located within the HindIII O fragment.

  18. The enigmatic genome of Chara australis virus.

    PubMed

    Gibbs, Adrian J; Torronen, Marjo; Mackenzie, Anne M; Wood, Jeffery T; Armstrong, John S; Kondo, Hideki; Tamada, Tetsuo; Keese, Paul L

    2011-11-01

    Most of the genomic sequence of Chara australis virus (CAV), previously called Chara corallina virus, has been determined. It is a ssRNA molecule of 9065 nt with at least four ORFs. At its 5' end is an ORF encoding a protein of 227 kDa, distantly homologous to the multifunctional replicases of benyviruses and rubiviruses. Next is an ORF encoding a protein of 44 kDa, homologous to the helicases of pestiviruses. The third ORF encodes an unmatched protein of 38 kDa that is probably a movement protein. The fourth and 3'-terminal ORF encodes a protein of 17.7 kDa homologous to the coat proteins of tobamoviruses. The short methyltransferase region of the CAV replicase matches only the C-terminal motif of benyvirus methyltransferases. This and other clues indicate that approximately 11% and 2% of the 5' and 3' termini of the complete CAV genome, respectively, are missing from the sequence. The aligned amino acid sequences of the CAV proteins and their nearest homologues contain many gaps but relationships inferred from them were little affected by removal of these gaps. Sequence comparisons show that three of the CAV genes may have diverged from the most closely related genes of other viruses 250-450 million years ago, and the sister relationship between the genes of CAV and those of benyviruses and tobamoviruses, mirroring the ancient sister relationship between charophytes (i.e. the algal host of CAV) and embryophytes (i.e. the plant hosts of tobamoviruses and benyviruses), is congruent with this possibility.

  19. Phosphorylation of ORF1p is required for L1 retrotransposition

    PubMed Central

    Cook, Pamela R.; Jones, Charles E.; Furano, Anthony V.

    2015-01-01

    Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1–host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host. PMID:25831499

  20. Phosphorylation of ORF1p is required for L1 retrotransposition.

    PubMed

    Cook, Pamela R; Jones, Charles E; Furano, Anthony V

    2015-04-01

    Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host. PMID:25831499

  1. Identification of a novel C16orf57 mutation in Athabaskan patients with Poikiloderma with Neutropenia.

    PubMed

    Clericuzio, Carol; Harutyunyan, Karine; Jin, Weidong; Erickson, Robert P; Irvine, Alan D; McLean, W H Irwin; Wen, Yaran; Bagatell, Rochelle; Griffin, Thomas A; Shwayder, Tor A; Plon, Sharon E; Wang, Lisa L

    2011-02-01

    Poikiloderma with Neutropenia (PN), Clericuzio-Type (OMIM #604173) is characterized by poikiloderma, chronic neutropenia, recurrent sinopulmonary infections, bronchiectasis, and nail dystrophy. First described by Clericuzio in 1991 in 14 patients of Navajo descent, it has since also been described in non-Navajo patients. C16orf57 has recently been identified as a causative gene in PN. The purpose of our study was to describe a spectrum of C16orf57 mutations in a cohort of PN patients including five patients of Athabaskan (Navajo and Apache) ancestry. Eleven patients from eight kindreds were enrolled in an IRB-approved study at Baylor College of Medicine. Five patients were of Athabaskan ancestry. PCR amplification and sequencing of the entire coding region of the C16orf57 gene was performed on genomic DNA. We identified biallelic C16orf57 mutations in all 11 PN patients in our cohort. The seven new deleterious mutations consisted of deletion (2), nonsense (3), and splice site (2) mutations. The patients of Athabaskan ancestry all had a common deletion mutation (c.496delA) which was not found in the six non-Athabaskan patients. Mutations in the C16orf57 gene have been identified thus far in all patients studied with a clinical diagnosis of PN. We have identified seven new mutations in C16orf57 in PN patients. One of these is present in all patients of Athabaskan descent, suggesting that c.496delA represents the PN-causative mutation in this subpopulation. PMID:21271650

  2. Ataxin-2 as potential disease modifier in C9ORF72 expansion carriers.

    PubMed

    van Blitterswijk, Marka; Mullen, Bianca; Heckman, Michael G; Baker, Matthew C; DeJesus-Hernandez, Mariely; Brown, Patricia H; Murray, Melissa E; Hsiung, Ging-Yuek R; Stewart, Heather; Karydas, Anna M; Finger, Elizabeth; Kertesz, Andrew; Bigio, Eileen H; Weintraub, Sandra; Mesulam, Marsel; Hatanpaa, Kimmo J; White, Charles L; Neumann, Manuela; Strong, Michael J; Beach, Thomas G; Wszolek, Zbigniew K; Lippa, Carol; Caselli, Richard; Petrucelli, Leonard; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Mackenzie, Ian R; Seeley, William W; Grinberg, Lea T; Miller, Bruce L; Boylan, Kevin B; Graff-Radford, Neill R; Boeve, Bradley F; Dickson, Dennis W; Rademakers, Rosa

    2014-10-01

    Repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are an important cause of both motor neuron disease (MND) and frontotemporal dementia (FTD). Currently, little is known about factors that could account for the phenotypic heterogeneity detected in C9ORF72 expansion carriers. In this study, we investigated 4 genes that could represent genetic modifiers: ataxin-2 (ATXN2), non-imprinted in Prader-Willi/Angelman syndrome 1 (NIPA1), survival motor neuron 1 (SMN1), and survival motor neuron 2 (SMN2). Assessment of these genes, in a unique cohort of 331 C9ORF72 expansion carriers and 376 control subjects, revealed that intermediate repeat lengths in ATXN2 possibly act as disease modifier in C9ORF72 expansion carriers; no evidence was provided for a potential role of NIPA1, SMN1, or SMN2. The effects of intermediate ATXN2 repeats were most profound in probands with MND or FTD/MND (2.1% vs. 0% in control subjects, p = 0.013), whereas the frequency in probands with FTD was identical to control subjects. Though intermediate ATXN2 repeats were already known to be associated with MND risk, previous reports did not focus on individuals with clear pathogenic mutations, such as repeat expansions in C9ORF72. Based on our present findings, we postulate that intermediate ATXN2 repeat lengths may render C9ORF72 expansion carriers more susceptible to the development of MND; further studies are needed, however, to validate our findings. PMID:24866401

  3. A Survey of Protein Structures from Archaeal Viruses

    PubMed Central

    Dellas, Nikki; Lawrence, C. Martin; Young, Mark J.

    2013-01-01

    Viruses that infect the third domain of life, Archaea, are a newly emerging field of interest. To date, all characterized archaeal viruses infect archaea that thrive in extreme conditions, such as halophilic, hyperthermophilic, and methanogenic environments. Viruses in general, especially those replicating in extreme environments, contain highly mosaic genomes with open reading frames (ORFs) whose sequences are often dissimilar to all other known ORFs. It has been estimated that approximately 85% of virally encoded ORFs do not match known sequences in the nucleic acid databases, and this percentage is even higher for archaeal viruses (typically 90%–100%). This statistic suggests that either virus genomes represent a larger segment of sequence space and/or that viruses encode genes of novel fold and/or function. Because the overall three-dimensional fold of a protein evolves more slowly than its sequence, efforts have been geared toward structural characterization of proteins encoded by archaeal viruses in order to gain insight into their potential functions. In this short review, we provide multiple examples where structural characterization of archaeal viral proteins has indeed provided significant functional and evolutionary insight. PMID:25371334

  4. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions

    PubMed Central

    Cooper-Knock, Johnathan; Walsh, Matthew J.; Higginbottom, Adrian; Robin Highley, J.; Dickman, Mark J.; Edbauer, Dieter; Ince, Paul G.; Wharton, Stephen B.; Wilson, Stuart A.; Kirby, Janine; Hautbergue, Guillaume M.

    2014-01-01

    GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72−) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry

  5. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions.

    PubMed

    Cooper-Knock, Johnathan; Walsh, Matthew J; Higginbottom, Adrian; Robin Highley, J; Dickman, Mark J; Edbauer, Dieter; Ince, Paul G; Wharton, Stephen B; Wilson, Stuart A; Kirby, Janine; Hautbergue, Guillaume M; Shaw, Pamela J

    2014-07-01

    GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72-) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry

  6. Identification of a family of group II introns encoding LAGLIDADG ORFs typical of group I introns.

    PubMed Central

    Toor, Navtej; Zimmerly, Steven

    2002-01-01

    Group I and group II introns are unrelated classes of introns that each encode proteins that facilitate intron splicing and intron mobility. Here we describe a new subfamily of nine introns in fungi that are group II introns but encode LAGLIDADG ORFs typical of group I introns. The introns have fairly standard group IIB1 RNA structures and are inserted into three different sites in SSU and LSU rRNA genes. Therefore, introns should not be assumed to be group I introns based solely on the presence of a LAGLIDADG ORF. PMID:12458791

  7. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

    Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  8. Hepatitis E rORF2p Stimulated and Unstimulated Peripheral Expression Profiling in Patients with Self-Limiting Hepatitis E Infection

    PubMed Central

    Rathod, Sanjay B.; Tripathy, Anuradha S.

    2014-01-01

    To improve the current knowledge on the involvement of peripheral lymphocytes in hepatitis E virus (HEV) associated pathogenesis, we analyzed alterations in (1) immunophenotypic expressions (by flow cytometry) and (2) gene expression patterns (by TaqMan Low Density Array) of activatory, inhibitory, integrin, homing, ectonucleotidase machinery, costimulatory, inflammatory markers, and T regulatory cells (Treg) associated cytokines on HEV rORF2p stimulated and unstimulated PBMCs of 43 acute HEV patients, 30 recovered individuals, and 43 controls. The phenotypic expressions of key molecules CTLA-4, GITR, CD103, CD25, CD69, IL10 and TGF-β1 in the acute patients and TGF-β1 in the recovered individuals were significantly elevated on both unstimulated and stimulated PBMCs. Gene expression array data revealed upregulations of CD25, PD1, CD103, CCR4, IL10, and TGF-β1 on both unstimulated and HEV rORF2p stimulated PBMCs of acute patients. The observed upregulations of inhibitory, integrin, activatory, and Treg-associated cytokine genes on the PBMCs of acute HEV patients complemented by their frequency data suggest them as the major players in the fine-tuning of immune response in self-limiting hepatitis E infection. PMID:24963498

  9. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets.

    PubMed

    Subissi, Lorenzo; Imbert, Isabelle; Ferron, François; Collet, Axelle; Coutard, Bruno; Decroly, Etienne; Canard, Bruno

    2014-01-01

    The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". PMID:24269475

  10. Expression of Amino-Terminal Portions or Full-Length Viral Replicase Genes in Transgenic Plants Confers Resistance to Potato Virus X Infection.

    PubMed Central

    Braun, CJ; Hemenway, CL

    1992-01-01

    The first open reading frame (ORF 1) of potato virus X (PVX) encodes a putative replicase gene. Transgenic tobacco lines expressing ORF 1 are resistant to PVX infection when inoculated with either PVX or PVX RNA. Analyses of lines containing various portions of the ORF 1 gene demonstrated that resistance is conferred to plants by expressing approximately the first half of the ORF 1 gene. One line expressing the untranslated leader and first 674 codons of ORF 1 is highly resistant to PVX infection. Conversely, lines expressing either approximately the third or fourth quarter of the ORF 1 gene, which contain the conserved nucleotide triphosphate (NTP) binding motif and Gly-Asp-Asp (GDD) motif, respectively, are not protected from PVX infection. In the resistant full-length and amino-terminal lines, lower numbers of local lesions were observed, and the virus accumulation in the inoculated and upper leaves was reduced when compared with the nontransformed control. When the performance of the most resistant ORF 1 line was compared with the most resistant coat protein (CP) line in a resistance test, the best ORF 1 line was more resistant to PVX infection than the best transgenic line expressing the PVX CP gene. These findings define a promising new approach for controlling plant viral infection. PMID:12297660

  11. C2ORF40 suppresses breast cancer cell proliferation and invasion through modulating expression of M phase cell cycle genes

    PubMed Central

    Lu, Jing; Wen, Mingxin; Huang, Yurong; He, Xiuquan; Wang, Yunshan; Wu, Qi; Li, Zengchun; Castellanos-Martin, Andres; Abad, Mar; Cruz-Hernandez, Juan J.; Rodriguez, Cesar A.; Perez-Losada, Jesus; Mao, Jian-Hua; Wei, Guangwei

    2013-01-01

    Recently, it has been suggested that C2ORF40 is a candidate tumor suppressor gene in breast cancer. However, the mechanism for reduced expression of C2ORF40 and its functional role in breast cancers remain unclear. Here we show that C2ORF40 is frequently silenced in human primary breast cancers and cell lines through promoter hypermethylation. C2ORF40 mRNA level is significantly associated with patient disease-free survival and distant cancer metastasis. Overexpression of C2ORF40 inhibits breast cancer cell proliferation, migration and invasion. By contrast, silencing C2ORF40 expression promotes these biological phenotypes. Bioinformatics and FACS analysis reveal C2ORF40 functions at G2/M phase by downregulation of mitotic genes expression, including UBE2C. Our results suggest that C2ORF40 acts as a tumor suppressor gene in breast cancer pathogenesis and progression and is a candidate prognostic marker for this disease. PMID:23770814

  12. Role of σ-factor (orf21) in clavulanic acid production in Streptomyces clavuligerus NRRL3585.

    PubMed

    Jnawali, Hum Nath; Liou, Kwangkyoung; Sohng, Jae Kyung

    2011-07-20

    A putative sigma factor gene, orf21, was disrupted or overexpressed in the wild-type clavulanic acid (CA) producer Streptomyces clavuligerus NRRL3585 and characterized. An orf21 mutant (Streptomyces clavuligerus HN14) of S. clavuligerus was obtained by insertional inactivation via double-crossover. Although there was little reduction of sporulation in the mutant, the growth pattern was similar between mutant and wild-type. The production was reduced by 10-15% in S. clavuligerus HN14 compared to that in wild-type. Overexpression of orf21 in wild-type cells caused hyperproduction of spores on solid medium and increased clavulanic acid production by 1.43-fold. The overexpression of orf21 in wild-type S. clavuligerus stimulated the expression of the early clavulanic acid genes, ceas2 and cas2, and the regulatory gene, ccaR, as demonstrated by RT-PCR. The elevation of the ceas2, cas2 and ccaR transcripts was consistent with the enhanced production of clavulanic acid.

  13. Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

    PubMed

    Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

    2015-05-01

    Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle. PMID:25973601

  14. Donkey orchid symptomless virus: a viral 'platypus' from Australian terrestrial orchids.

    PubMed

    Wylie, Stephen J; Li, Hua; Jones, Michael G K

    2013-01-01

    Complete and partial genome sequences of two isolates of an unusual new plant virus, designated Donkey orchid symptomless virus (DOSV) were identified using a high-throughput sequencing approach. The virus was identified from asymptomatic plants of Australian terrestrial orchid Diuris longifolia (Common donkey orchid) growing in a remnant forest patch near Perth, western Australia. DOSV was identified from two D. longifolia plants of 264 tested, and from at least one plant of 129 Caladenia latifolia (pink fairy orchid) plants tested. Phylogenetic analysis of the genome revealed open reading frames (ORF) encoding seven putative proteins of apparently disparate origins. A 69-kDa protein (ORF1) that overlapped the replicase shared low identity with MPs of plant tymoviruses (Tymoviridae). A 157-kDa replicase (ORF2) and 22-kDa coat protein (ORF4) shared 32% and 40% amino acid identity, respectively, with homologous proteins encoded by members of the plant virus family Alphaflexiviridae. A 44-kDa protein (ORF3) shared low identity with myosin and an autophagy protein from Squirrelpox virus. A 27-kDa protein (ORF5) shared no identity with described proteins. A 14-kDa protein (ORF6) shared limited sequence identity (26%) over a limited region of the envelope glycoprotein precursor of mammal-infecting Crimea-Congo hemorrhagic fever virus (Bunyaviridae). The putative 25-kDa movement protein (MP) (ORF7) shared limited (27%) identity with 3A-like MPs of members of the plant-infecting Tombusviridae and Virgaviridae. Transmissibility was shown when DOSV systemically infected Nicotiana benthamiana plants. Structure and organization of the domains within the putative replicase of DOSV suggests a common evolutionary origin with 'potexvirus-like' replicases of viruses within the Alphaflexiviridae and Tymoviridae, and the CP appears to be ancestral to CPs of allexiviruses (Alphaflexiviridae). The MP shares an evolutionary history with MPs of dianthoviruses, but the other putative

  15. ORF13 in the Type III secretion system gene cluster of Edwardsiella tarda binds to the mammalian factor Cugbp2.

    PubMed

    Okuda, Jun; Takeuchi, Yusuke; Yasuda, Masashi; Nakai, Toshihiro

    2016-05-01

    The Type III secretion system (TTSS) is essential for the intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals, and a hypothetical gene (orf13) located in the TTSS gene cluster is required for intracellular replication and virulence of E. tarda. Here, we show that under TTSS-inducing conditions, the protein ORF13 was secreted into culture supernatant. Then, using a yeast 2-hybrid screen, we show that the mammalian factor Cugbp2, which regulates apoptosis in breast cancer cells, directly interacts with ORF13. A pull-down assay revealed that ORF13 binds to the C-terminal region of Cugbp2. Our results suggest that ORF13 may facilitate E. tarda replication in phagocytes by binding to Cugbp2. PMID:27137075

  16. C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice

    PubMed Central

    Atanasio, Amanda; Decman, Vilma; White, Derek; Ramos, Meg; Ikiz, Burcin; Lee, Hoi-Ching; Siao, Chia-Jen; Brydges, Susannah; LaRosa, Elizabeth; Bai, Yu; Fury, Wen; Burfeind, Patricia; Zamfirova, Ralica; Warshaw, Gregg; Orengo, Jamie; Oyejide, Adelekan; Fralish, Michael; Auerbach, Wojtek; Poueymirou, William; Freudenberg, Jan; Gong, Guochun; Zambrowicz, Brian; Valenzuela, David; Yancopoulos, George; Murphy, Andrew; Thurston, Gavin; Lai, Ka-Man Venus

    2016-01-01

    The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology. PMID:26979938

  17. Novel hepatitis E like virus found in Swedish moose.

    PubMed

    Lin, Jay; Norder, Heléne; Uhlhorn, Henrik; Belák, Sándor; Widén, Frederik

    2014-03-01

    A novel virus was detected in a sample collected from a Swedish moose (Alces alces). The virus was suggested as a member of the Hepeviridae family, although it was found to be highly divergent from the known four genotypes (gt1-4) of hepatitis E virus (HEV). Moose are regularly hunted for consumption in the whole of Scandinavia. Thus, the finding of this virus may be important from several aspects: (a) as a new diverged HEV in a new animal species, and (b) potential unexplored HEV transmission pathways for human infections. Considering these aspects, we have started the molecular characterization of this virus. A 5.1 kb amplicon was sequenced, and corresponded to the partial ORF1, followed by complete ORF2, ORF3 and poly(A) sequence. In comparison with existing HEVs, the moose HEV genome showed a general nucleotide sequence similarity of 37-63% and an extensively divergent putative ORF3 sequence. The junction region between the ORFs was also highly divergent; however, two putative secondary stem-loop structures were retained when compared to gt1-4, but with altered structural appearance. In the phylogenetic analysis, the moose HEV deviated and formed its own branch between the gt1-4 and other divergent animal HEVs. The characterization of this highly divergent genome provides important information regarding the diversity of HEV infecting various mammalian species. However, further studies are needed to investigate its prevalence in the moose populations and possibly in other host species, including the risk for human infection.

  18. Depletion of C3orf1/TIMMDC1 inhibits migration and proliferation in 95D lung carcinoma cells.

    PubMed

    Wu, Huiling; Wang, Wenbing; Xu, Huaxi

    2014-01-01

    In our previous study, we identified an association of high expression of c3orf1, also known as TIMMDC1 (translocase of inner mitochondrial membrane domain-containing protein 1), with metastatic characteristics in lung carcinoma cells. To investigate the preliminary function and mechanism of this mitochondrial protein, we depleted C3orf1 expression by introducing siRNA into 95D lung carcinoma cells. We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration. We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1. Microarray data indicated that genes involved in regulation of cell death, migration, and cell-cycle arrest were significantly altered after C3orf1 depletion for 48 h. The expression of genes involved in focal adhesion, ECM-receptor interaction, and p53-signaling pathways were notably altered. Furthermore, cell-cycle arrest genes such as CCNG2 and PTEN as well as genes involved in cell migration inhibition, such as TIMP3 and COL3A1, were upregulated after C3orf1 depletion in 95D cells. Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR. We conclude that C3orf1 is critical for mitochondrial function, migration, and proliferation in 95D lung carcinoma cells. Depletion of C3orf1 inhibited cell migration and cell proliferation in association with upregulation of genes involved in cell-cycle arrest and cell migration inhibition. These results suggest that C3orf1 (TIMMDC1) may be a viable treatment target for lung carcinoma, and that further study of the role of this protein in lung carcinoma pathogenesis is justified. PMID:25391042

  19. Contagious Ecthyma, Rangiferine Brucellosis, and Lungworm Infection in a Muskox ( Ovibos moschatus ) from the Canadian Arctic, 2014.

    PubMed

    Tomaselli, Matilde; Dalton, Chimoné; Duignan, Pádraig J; Kutz, Susan; van der Meer, Frank; Kafle, Pratap; Surujballi, Om; Turcotte, Claude; Checkley, Sylvia

    2016-07-01

    An adult male muskox ( Ovibos moschatus ), harvested on 26 August 2014 on Victoria Island, Nunavut, in the Canadian Arctic, had proliferative dermatitis on the muzzle and fetlocks suggestive of contagious ecthyma or orf (Parapoxvirus). Histopathologic features of the lesions were consistent with this diagnosis. Orf virus DNA, phylogenetically similar to an isolate from a captive muskox of the Minnesota Zoo, US, was detected in the lesions by PCR using Parapoxvirus primers. Additionally, there was a metaphyseal abscess with a cortical fistula in the right metacarpus from which Brucella suis biovar 4 was isolated and identification supported by PCR. Brucella spp. antibodies were detected in serum. Finally, 212 nodules were dissected from the lungs. Fecal analysis and lung examination demonstrated co-infection with the lungworms Umingmakstrongylus pallikuukensis and Varestrongylus eleguneniensis. The zoonotic potential of orf and rangiferine brucellosis adds an important public health dimension to this case, particularly given that muskoxen are a valuable source of food for Arctic residents. Careful examination of these pathogens at a population level is needed as they may contribute to muskox population decline and potentially constitute a driver of food insecurity for local communities. This case underscores the importance of wildlife health surveillance as a management tool to conserve wildlife populations and maintain food security in subsistence-oriented communities.

  20. Contagious Ecthyma, Rangiferine Brucellosis, and Lungworm Infection in a Muskox ( Ovibos moschatus ) from the Canadian Arctic, 2014.

    PubMed

    Tomaselli, Matilde; Dalton, Chimoné; Duignan, Pádraig J; Kutz, Susan; van der Meer, Frank; Kafle, Pratap; Surujballi, Om; Turcotte, Claude; Checkley, Sylvia

    2016-07-01

    An adult male muskox ( Ovibos moschatus ), harvested on 26 August 2014 on Victoria Island, Nunavut, in the Canadian Arctic, had proliferative dermatitis on the muzzle and fetlocks suggestive of contagious ecthyma or orf (Parapoxvirus). Histopathologic features of the lesions were consistent with this diagnosis. Orf virus DNA, phylogenetically similar to an isolate from a captive muskox of the Minnesota Zoo, US, was detected in the lesions by PCR using Parapoxvirus primers. Additionally, there was a metaphyseal abscess with a cortical fistula in the right metacarpus from which Brucella suis biovar 4 was isolated and identification supported by PCR. Brucella spp. antibodies were detected in serum. Finally, 212 nodules were dissected from the lungs. Fecal analysis and lung examination demonstrated co-infection with the lungworms Umingmakstrongylus pallikuukensis and Varestrongylus eleguneniensis. The zoonotic potential of orf and rangiferine brucellosis adds an important public health dimension to this case, particularly given that muskoxen are a valuable source of food for Arctic residents. Careful examination of these pathogens at a population level is needed as they may contribute to muskox population decline and potentially constitute a driver of food insecurity for local communities. This case underscores the importance of wildlife health surveillance as a management tool to conserve wildlife populations and maintain food security in subsistence-oriented communities. PMID:27285415

  1. A variant of Rubus yellow net virus with altered genomic organization.

    PubMed

    Diaz-Lara, Alfredo; Mosier, Nola J; Keller, Karen E; Martin, Robert R

    2015-02-01

    Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS.

  2. A variant of Rubus yellow net virus with altered genomic organization.

    PubMed

    Diaz-Lara, Alfredo; Mosier, Nola J; Keller, Karen E; Martin, Robert R

    2015-02-01

    Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS. PMID:25480633

  3. Requirement of UAP56, URH49, RBM15, and OTT3 in the expression of Kaposi sarcoma-associated herpesvirus ORF57

    SciTech Connect

    Majerciak, Vladimir; Deng, Merlyn; Zheng Zhiming

    2010-11-25

    Transport of mRNA from the nucleus to the cytoplasm is mediated by cellular RNA export factors. In this report, we examined how RNA export factors UAP56 and URH49, and RNA export cofactors RBM15 and OTT3, function in modulating KSHV ORF57 expression. We found that knockdown of each factor by RNAi led to decreased ORF57 expression. Specifically, reduced expression of either UAP56 or RBM15 led to nuclear export deficiency of ORF57 RNA. In the context of the KSHV genome, the near absence of UAP56 or RBM15 reduced the expression of both ORF57 and ORF59 (an RNA target of ORF57), but not ORF50. Collectively, our data indicate that the expression of KSHV ORF57 is regulated by cellular RNA export factors and cofactors at the posttranscriptional level.

  4. Sequencing of the bicistronic genome segments S7 and S9 of Mal de Río Cuarto virus (Fijivirus, Reoviridae) completes the genome of this virus.

    PubMed

    Guzmán, F A; Distéfano, A J; Arneodo, J D; Hopp, H E; Lenardon, S L; del Vas, M; Conci, L R

    2007-01-01

    The nucleotide sequences of genomic segments S7 and S9 of Mal de Río Cuarto virus (MRCV, Fijivirus group II) have been determined, thus completing the entire genome sequence of the virus. These segments showed a non-overlapping bicistronic structure, as in other members of the genus. MRCV S7 ORF-1 had a length of 1086 bp and encoded a 41.5 kDa putative polypeptide, whereas MRCV S7 ORF-2 had a length of 930 bp and encoded a 36.8 kDa putative polypeptide. Proteins of 39 and 20.5 kDa were predicted for the 1014 bp long MRCV S9 ORF-1 and the 537 bp long MRCV S9 ORF-2, respectively. The terminal 5' and 3' sequences of both segments were 5'AAGUUUUU3' and 5'CAGCUnnnGUC3', respectively. Specific imperfect inverted repeats of each segment were identified. Comparison of the predicted proteins with those of related virus genome segments counterparts in maize rough dwarf virus (MRDV) and rice black streaked dwarf virus (RBSDV), showed 64.5-44.3% identities. These values are lower than those resulting from comparisons between MRDV and RBSDV. The topology of the trees obtained using the complete nucleotide and amino acid sequences of MRCV S7 and MRCV S9 was consistent with the analysis of the other MRCV segments previously published.

  5. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed Central

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-01-01

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region. PMID:3671088

  6. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-10-26

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region.

  7. orf4 of the Bacillus cereus sigB gene cluster encodes a general stress-inducible Dps-like bacterioferritin.

    PubMed

    Wang, Shin-Wei; Chen, Chien-Yen; Tseng, Joseph T; Liang, Shih-Hsiung; Chen, Ssu-Ching; Hsieh, Chienyan; Chen, Yen-hsu; Chen, Chien-Cheng

    2009-07-01

    The function of orf4 in the sigB cluster in Bacillus cereus ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an orf4-null mutant and produced recombinant protein rOrf4 to establish the role of orf4. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl(2). Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of orf4 had no effect on activation of the alternative sigma factor sigma(B), and therefore, orf4 is not associated with sigma(B) regulation; however, orf4 can be significantly upregulated upon environmental stress but not H(2)O(2) treatment. B. cereus strains with constitutive Orf4 expression exhibited a viability higher than that of the orf4-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding. PMID:19429618

  8. Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

    PubMed

    Micci, Francesca; Panagopoulos, Ioannis; Thorsen, Jim; Davidson, Ben; Tropé, Claes Gøran; Heim, Sverre

    2014-02-01

    The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15%) serous ovarian carcinomas examined, a finding that holds great promise for our understanding of ovarian tumorigenesis as well as, potentially, for new treatment strategies. We wanted to test how frequent the ESRRA/C11orf20 fusion is in ovarian carcinomas of all subtypes, and therefore examined a series of 230 ovarian carcinomas of which 197 were of the serous subtype and 163 of the 197 were of stages III and IV--that is, the very same carcinoma subset where the fusion transcript had been found. We performed PCR and high-throughput sequencing analyses in search of the fusion transcript. We used the same primers described previously for the detection of the fusion and the same primer combination, but found no ESRRA/C11orf20 fusion in our series. A synthetic DNA plasmid containing the reported ESRRA/C11orf20 fusion was included as a positive control for our PCR experiments. Data from high-throughput sequencing of 23 ovarian carcinomas were screened in search of alternative partner(s) for the ESRRA and/or C11orf20 gene, but none was found. We conclude that the frequency of the ESRRA/C11orf20 gene fusion in serous ovarian carcinomas of stages III and IV must be considerable less than that reported previously (0/163 in our experience compared with 10/67 in the previous study). At the very least, it seems clear that the said fusion cannot be a common pathogenetic event in this tumor type.

  9. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  10. A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy

    PubMed Central

    Yang, Mei; Liang, Chen; Swaminathan, Kunchithapadam; Herrlinger, Stephanie; Lai, Fan; Shiekhattar, Ramin; Chen, Jian-Fu

    2016-01-01

    The intronic GGGGCC hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is a prevalent genetic abnormality identified in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) is a protein with unclear functions. We report that C9ORF72 is a component of a multiprotein complex containing SMCR8, WDR41, and ATG101 (an important regulator of autophagy). The C9ORF72 complex displays guanosine triphosphatase (GTPase) activity and acts as a guanosine diphosphate–guanosine 5′-triphosphate (GDP-GTP) exchange factor (GEF) for RAB39B. We created Smcr8 knockout mice and found that Smcr8 mutant cells exhibit impaired autophagy induction, which is similarly observed in C9orf72 knockdown cells. Mechanistically, SMCR8/C9ORF72 interacts with the key autophagy initiation ULK1 complex and regulates expression and activity of ULK1. The complex has an additional role in regulating later stages of autophagy. Whereas autophagic flux is enhanced in C9orf72 knockdown cells, depletion of Smcr8 results in a reduced flux with an abnormal expression of lysosomal enzymes. Thus, C9ORF72 and SMCR8 have similar functions in modulating autophagy induction by regulating ULK1 and play distinct roles in regulating autophagic flux.

  11. A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy

    PubMed Central

    Yang, Mei; Liang, Chen; Swaminathan, Kunchithapadam; Herrlinger, Stephanie; Lai, Fan; Shiekhattar, Ramin; Chen, Jian-Fu

    2016-01-01

    The intronic GGGGCC hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is a prevalent genetic abnormality identified in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) is a protein with unclear functions. We report that C9ORF72 is a component of a multiprotein complex containing SMCR8, WDR41, and ATG101 (an important regulator of autophagy). The C9ORF72 complex displays guanosine triphosphatase (GTPase) activity and acts as a guanosine diphosphate–guanosine 5′-triphosphate (GDP-GTP) exchange factor (GEF) for RAB39B. We created Smcr8 knockout mice and found that Smcr8 mutant cells exhibit impaired autophagy induction, which is similarly observed in C9orf72 knockdown cells. Mechanistically, SMCR8/C9ORF72 interacts with the key autophagy initiation ULK1 complex and regulates expression and activity of ULK1. The complex has an additional role in regulating later stages of autophagy. Whereas autophagic flux is enhanced in C9orf72 knockdown cells, depletion of Smcr8 results in a reduced flux with an abnormal expression of lysosomal enzymes. Thus, C9ORF72 and SMCR8 have similar functions in modulating autophagy induction by regulating ULK1 and play distinct roles in regulating autophagic flux. PMID:27617292

  12. CSBF/C10orf99, a novel potential cytokine, inhibits colon cancer cell growth through inducing G1 arrest.

    PubMed

    Pan, Wen; Cheng, Yingying; Zhang, Heyu; Liu, Baocai; Mo, Xiaoning; Li, Ting; Li, Lin; Cheng, Xiaojing; Zhang, Lianhai; Ji, Jiafu; Wang, Pingzhang; Han, Wenling

    2014-01-01

    Cytokines are soluble proteins that exert their functions by binding specific receptors. Many cytokines play essential roles in carcinogenesis and have been developed for the treatment of cancer. In this study, we identified a novel potential cytokine using immunogenomics designated colon-derived SUSD2 binding factor (CSBF), also known as chromosome 10 open reading frame 99 (C10orf99). CSBF/C10orf99 is a classical secreted protein with predicted molecular mass of 6.5 kDa, and a functional ligand of Sushi Domain Containing 2 (SUSD2). CSBF/C10orf99 has the highest expression level in colon tissue. Both CSBF/C10orf99 and SUSD2 are down-regulated in colon cancer tissues and cell lines with different regulation mechanisms. CSBF/C10orf99 interacts with SUSD2 to inhibit colon cancer cell growth and induce G1 cell cycle arrest by down-regulating cyclin D and cyclin-dependent kinase 6 (CDK6). CSBF/C10orf99 displays a bell-shaped activity curve with the optimal effect at ~10 ng/ml. Its growth inhibitory effects can be blocked by sSUSD2-Fc soluble protein. Our results suggest that CSBF/C10orf99 is a novel potential cytokine with tumor suppressor functions. PMID:25351403

  13. Distinct clinical and pathological phenotypes in frontotemporal dementia associated with MAPT, PGRN and C9orf72 mutations.

    PubMed

    Snowden, Julie S; Adams, Jennifer; Harris, Jennifer; Thompson, Jennifer C; Rollinson, Sara; Richardson, Anna; Jones, Matthew; Neary, David; Mann, David M; Pickering-Brown, Stuart

    2015-01-01

    Our objective was to compare the clinical and pathological characteristics of frontotemporal dementia patients with MAPT, GRN and C9orf72 gene mutations. We carried out a cross-sectional comparative study of 74 gene-positive patients (15 MAPT, 17 GRN and 42 C9orf72). Thirty had post mortem pathological data permitting clinico-pathological correlation. MAPT patients were younger than other groups, and showed more frequent behavioural disinhibition, repetitive and stereotyped behaviours, semantic impairment and temporal predominance of atrophy. GRN patients were older at death and more likely to present with non-fluent aphasia. C9orf72 patients alone showed a co-occurrence of ALS. They showed more psychotic symptoms and irrational behaviour, yet were more often reported clinically as socially appropriate and warm. They showed less dietary change than other groups. C9orf72 patients with and without ALS differed only in frequency of psychosis. Greater clinical overlap was observed between GRN and C9orf72 compared to MAPT cases. MAPT cases had tau and GRN and C9orf72, with one exception, TDP-43 pathology. Non-fluent aphasia was linked to TDP subtype A in both GRN and C9orf72 cases and ALS with subtype B. In conclusion, the findings reinforce clinical heterogeneity in FTD and strengthen evidence that genotype influences clinical presentation. Clinical features may inform targeted genetic testing. PMID:26473392

  14. A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy.

    PubMed

    Yang, Mei; Liang, Chen; Swaminathan, Kunchithapadam; Herrlinger, Stephanie; Lai, Fan; Shiekhattar, Ramin; Chen, Jian-Fu

    2016-09-01

    The intronic GGGGCC hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is a prevalent genetic abnormality identified in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) is a protein with unclear functions. We report that C9ORF72 is a component of a multiprotein complex containing SMCR8, WDR41, and ATG101 (an important regulator of autophagy). The C9ORF72 complex displays guanosine triphosphatase (GTPase) activity and acts as a guanosine diphosphate-guanosine 5'-triphosphate (GDP-GTP) exchange factor (GEF) for RAB39B. We created Smcr8 knockout mice and found that Smcr8 mutant cells exhibit impaired autophagy induction, which is similarly observed in C9orf72 knockdown cells. Mechanistically, SMCR8/C9ORF72 interacts with the key autophagy initiation ULK1 complex and regulates expression and activity of ULK1. The complex has an additional role in regulating later stages of autophagy. Whereas autophagic flux is enhanced in C9orf72 knockdown cells, depletion of Smcr8 results in a reduced flux with an abnormal expression of lysosomal enzymes. Thus, C9ORF72 and SMCR8 have similar functions in modulating autophagy induction by regulating ULK1 and play distinct roles in regulating autophagic flux. PMID:27617292

  15. RFHVMn ORF73 is structurally related to the KSHV ORF73 latency-associated nuclear antigen (LANA) and is expressed in retroperitoneal fibromatosis (RF) tumor cells

    SciTech Connect

    Burnside, Kellie L.; Ryan, Jonathan T.; Bielefeldt-Ohmann, Helle; Gregory Bruce, A.; Thouless, Margaret E.; Tsai, Che-Chung; Rose, Timothy M. . E-mail: trose@u.washington.edu

    2006-10-10

    Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), was first identified in retroperitoneal fibromatosis (RF) tumor lesions of macaques with simian AIDS. We cloned and sequenced the ORF73 latency-associated nuclear antigen (LANA) of RFHVMn from the pig-tailed macaque. RFHVMn LANA is structurally analogous to KSHV ORF73 LANA and contains an N-terminal serine-proline-rich region, a large internal glutamic acidic-rich repeat region and a conserved C-terminal domain. RFHVMn LANA reacts with monoclonal antibodies specific for a glutamic acid-proline dipeptide motif and a glutamic acid-glutamine-rich motif in the KSHV LANA repeat region. Immunohistochemical and immunofluorescence analysis revealed that RFHVMn LANA is a nuclear antigen which is highly expressed in RF spindloid tumor cells. These data suggest that RFHV LANA is an ortholog of KSHV LANA and will function similarly to maintain viral latency and play a role in tumorigenicity in macaques.

  16. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

    PubMed

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

    2014-11-01

    Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

  17. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

    PubMed Central

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

    2014-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

  18. Characterization of specific antigenic epitopes and the nuclear export signal of the Porcine circovirus 2 ORF3 protein.

    PubMed

    Gu, Jinyan; Wang, Lun; Jin, Yulan; Lin, Cui; Wang, Huijuan; Zhou, Niu; Xing, Gang; Liao, Min; Zhou, Jiyong

    2016-02-29

    Porcine circovirus 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome. PCV2 ORF3 protein is a nonstructural protein known to induce apoptosis, but little is known about the biological function of ORF3 protein. Therefore, we undertook this study to map ORF3 protein epitopes recognized by a panel of monoclonal antibodies (mAbs) and to characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3. The linear epitopes targeted by two previously published mAbs 3B1 and 1H3 and a novel mouse mAb 3C3 were defined using overlapping pools of peptides. Here, we find that ORF3 in PCV2 infected cells contains a conformational epitope targeted by the antibody 3C3, which is distinct from linear epitopes recognized by the antibodies 3B1 and 1H3 in recombinant ORF3 protein. These results suggest that the linear epitope recognized by 3B1 and 1H3 is masked in PCV2 infected cells, and that the conformational epitope is unique to PCV2 infection. Furthermore, we find that ORF3 protein expressed in cytoplasm in early stages of PCV2 infection and then accumulated in nucleus over time. Moreover, we localize a NES at the N-terminus (residues 1-35aa) of ORF3 which plays critical role in nuclear export activity. These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3. PMID:26854343

  19. Deep sequencing reveals the complete genome and evidence for transcriptional activity of the first virus-like sequences identified in Aristotelia chilensis (Maqui Berry).

    PubMed

    Villacreses, Javier; Rojas-Herrera, Marcelo; Sánchez, Carolina; Hewstone, Nicole; Undurraga, Soledad F; Alzate, Juan F; Manque, Patricio; Maracaja-Coutinho, Vinicius; Polanco, Victor

    2015-04-01

    Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%-73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant. PMID:25855242

  20. Deep Sequencing Reveals the Complete Genome and Evidence for Transcriptional Activity of the First Virus-Like Sequences Identified in Aristotelia chilensis (Maqui Berry)

    PubMed Central

    Villacreses, Javier; Rojas-Herrera, Marcelo; Sánchez, Carolina; Hewstone, Nicole; Undurraga, Soledad F.; Alzate, Juan F.; Manque, Patricio; Maracaja-Coutinho, Vinicius; Polanco, Victor

    2015-01-01

    Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant. PMID:25855242

  1. Discovery of Human sORF-Encoded Polypeptides (SEPs) in Cell Lines and Tissue

    PubMed Central

    2015-01-01

    The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation. PMID:24490786

  2. C9ORF72 hexanucleotide repeat expansion in ALS patients from the Central European Russia population.

    PubMed

    Abramycheva, Natalya Y; Lysogorskaia, Elena V; Stepanova, Maria S; Zakharova, Maria N; Kovrazhkina, Elena A; Razinskaya, Olga D; Smirnov, Andrey P; Maltsev, Andrey V; Ustyugov, Alexey A; Kukharsky, Michail S; Khritankova, Inna V; Bachurin, Sergey O; Cooper-Knock, Johnathan; Buchman, Vladimir L; Illarioshkin, Sergey N; Skvortsova, Veronika I; Ninkina, Natalia

    2015-10-01

    Cohorts of amyotrophic lateral sclerosis (ALS) patients and control individuals of Caucasian origin from the Central European Russia (Moscow city and region) were analyzed for the presence of hexanucleotide repeat GGGGCC expansion within the first intron of the C9ORF72 gene. The presence of a large (>40) repeat expansion was found in 15% of familial ALS cases (3 of 20 unrelated familial cases) and 2.5% of sporadic ALS cases (6 of 238) but in none of control cases. These results suggest that the frequency of C9ORF72 hexanucleotide repeats expansions in the Central European Russian ALS patients is significantly lower than in Western European or Northern American ALS patients of Caucasian origin but higher than in Asian ALS patients.

  3. A stable RNA virus-based vector for citrus trees

    SciTech Connect

    Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe; Dawson, William O.

    2007-11-10

    Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.

  4. Atypical, slowly progressive behavioral variant frontotemporal dementia associated with C9ORF72 hexanucleotide expansion

    PubMed Central

    Khan, Baber K.; Yokoyama, Jennifer S.; Takada, Leonel T.; Sha, Sharon J.; Rutherford, Nicola. J.; Fong, Jamie C.; Karydas, Anna; Wu, Teresa; Ketelle, Robin; Baker, Matt C.; Hernandez, Mariely-Dejesus; Coppola, Giovanni; Geschwind, Daniel H.; Rademakers, Rosa; Lee, Suzee E.; Rosen, Howard J.; Rabinovici, Gil D.; Seeley, William; Rankin, Katherine P.; Boxer, Adam L.; Miller, Bruce L.

    2012-01-01

    Background Some patients meeting behavioral variant frontotemporal dementia (bvFTD) diagnostic criteria progress slowly and plateau at mild symptom severity. Such patients have mild neuropsychological and functional impairments, lack characteristic bvFTD brain atrophy, and have thus been referred to as bvFTD “phenocopies” or slowly progressive (bvFTD-SP). The few patients with bvFTD-SP that have been studied at autopsy have found no evidence of FTD pathology, suggesting that bvFTD-SP is neuropathologically distinct from other forms of FTD. Here, we describe two patients with bvFTD-SP with chromosome 9 open reading frame 72 (C9ORF72) hexanucleotide expansions. Methods Three hundred and eighty-four patients with FTD clinical spectrum and Alzheimer’s disease diagnoses were screened for C9ORF72 expansion. Two bvFTD-SP mutation carriers were identified. Neuropsychological and functional data, as well as brain atrophy patterns assessed using voxel-based morphometry (VBM), were compared with 44 patients with sporadic bvFTD and 85 healthy controls. Results Both patients were age 48 at baseline and met possible bvFTD criteria. In the first patient, VBM revealed thalamic and posterior insula atrophy. Over seven years, his neuropsychological performance and brain atrophy remained stable. In the second patient, VBM revealed cortical atrophy with subtle frontal and insular volume loss. Over two years, her neuropsychological and functional scores as well as brain atrophy remained stable. Conclusions C9ORF72 mutations can present with a bvFTD-SP phenotype. Some bvFTD-SP patients may have neurodegenerative pathology, and C9ORF72 mutations should be considered in patients with bvFTD-SP and a family history of dementia or motor neuron disease. PMID:22399793

  5. C9orf72 Hexanucleotide Repeat Expansion and Guam Amyotrophic Lateral Sclerosis–Parkinsonism-Dementia Complex

    PubMed Central

    Dombroski, Beth A.; Galasko, Douglas R.; Mata, Ignacio F.; Zabetian, Cyrus P.; Craig, Ulla-Katrina; Garruto, Ralph M.; Oyanagi, Kiyomitsu; Schellenberg, Gerard D.

    2013-01-01

    Importance High-prevalence foci of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) exist in Japanese on the Kii Peninsula of Japan and in the Chamorros of Guam. Clinical and neuropathologic similarities suggest that the disease in these 2 populations may be related. Recent findings showed that some of the Kii Peninsula ALS cases had pathogenic C9orf72 repeat expansions, a genotype that causes ALS in Western populations. Objectives To perform genotyping among Guam residents to determine if the C9orf72 expanded repeat allele contributes to ALS-PDC in this population and to evaluate LRRK2 for mutations in the same population. Design and Setting Case-control series from neurodegenerative disease research programs on Guam that screened residents for ALS, PDC, and dementia. Participants Study participants included 24 with ALS and 22 with PDC and 43 older control subjects with normal cognition ascertained between 1956 and 2006. All but one participant were Chamorro, the indigenous people of Guam. A single individual of white race/ethnicity with ALS was ascertained on Guam during the study. Main Outcomes and Measures Participants were screened for C9orf72 hexanucleotide repeat length. Participants with repeat numbers in great excess of 30 were considered to have pathogenic repeat expansions. LRRK2 was screened for point mutations by DNA sequencing. Results We found a single individual with an expanded pathogenic hexanucleotide repeat. This individual of white race/ethnicity with ALS was living on Guam at the time of ascertainment but had been born in the United States. All Chamorro participants with ALS and PDC and control subjects had normal repeats, ranging from 2 to 17 copies. No pathogenic LRRK2 mutations were found. Conclusions and Relevance Unlike participants with ALS from the Kii Peninsula, C9orf72 expansions do not cause ALS-PDC in Chamorros. Likewise, LRRK2 mutations do not cause Guam ALS-PDC. PMID:23588498

  6. Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.

    PubMed

    Evans, Iona C; Barnes, Josephine L; Garner, Ian M; Pearce, David R; Maher, Toby M; Shiwen, Xu; Renzoni, Elisabetta A; Wells, Athol U; Denton, Christopher P; Laurent, Geoffrey J; Abraham, David J; McAnulty, Robin J

    2016-04-01

    Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

  7. Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.

    PubMed

    Barbagallo, Michael S; Birch, Katherine E; Deacon, Nicholas J; Mosse, Jennifer A

    2012-07-01

    The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.

  8. DNA sequence analysis of the Hind III M fragment from Chinese vaccine strain of vaccinia virus.

    PubMed

    Liu, V J; Jin, Q; Jin, D Y; Hou, Y D

    1989-01-01

    The complete DNA sequence of the Hind III M fragment of vaccinia virus (VV) Tian Tan strain genome was determined by the dideoxynucleotide chain termination method. Three open reading frames (ORFs) were identified in the complementary strand of the sequence, comprised of 2218bp. Among them, ORF K1 initiates its transcription at -45 of the Hind III K fragment. The deduced peptide encoded by K1 contains 284 amino acids with a calculated molecular weight of 32.48 KDa. Its sequence is homologous to the host range protein of VV Copenhagen strain; the variation is only 2.46% at the amino acid level. ORF M2 could encode a peptide of 21.94 KDa with 196 amino acids. This gene was shown to be homologous to that of the 23 KDa peptide of herpes simplex virus type I. A non-coding region of 204bp located between K1 and M2 is rich in palindromic structures. ORF M1 extends its 3' terminus into the Hind III N fragment. Within the M fragment, M1 can only encode 212 amino acids. The major part of ORF M1 is very similar to the M portion of a possible alpha-amanitin resistance gene isolated from VV-WR strain. This work provides a molecular foundation in the construction of a new insertion vector for the preparation of a recombinant vaccinia virus to be used as a polyvalent live vaccine.

  9. Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter.

    PubMed

    Man, Michal; Epel, Bernard L

    2004-06-01

    A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

  10. Discovery of protein interactions using parallel analysis of translated ORFs (PLATO).

    PubMed

    Larman, H Benjamin; Liang, Anthony C; Elledge, Stephen J; Zhu, Jian

    2014-01-01

    Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d. PMID:24336473

  11. Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus.

    PubMed

    Kazmi, Syeda Amber; Yang, Zuokun; Hong, Ni; Wang, Guoping; Wang, Yanfen

    2015-01-01

    RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region.

  12. Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus

    PubMed Central

    Kazmi, Syeda Amber; Yang, Zuokun; Hong, Ni; Wang, Guoping; Wang, Yanfen

    2015-01-01

    RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region. PMID:26207896

  13. Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus.

    PubMed

    Kazmi, Syeda Amber; Yang, Zuokun; Hong, Ni; Wang, Guoping; Wang, Yanfen

    2015-01-01

    RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region. PMID:26207896

  14. Opium poppy mosaic virus, a new umbravirus isolated from Papaver somniferum in New Zealand.

    PubMed

    Tang, Joe; Lebas, Bénédicte; Liefting, Lia; Veerakone, Stella; Wei, Ting; Ward, Lisa

    2016-01-01

    A novel virus, tentatively named "opium poppy mosaic virus" (OPMV), was isolated from Papaver somniferum (opium poppy) with leaf mosaic and mottling symptoms in Auckland, New Zealand, in 2006. The virus was mechanically transmitted to herbaceous plants of several species, in which it induced local and/or systemic symptoms. No virus particles were observed by electron microscopy in the diseased P. somniferum or any of the symptomatic herbaceous plants. The complete genomic sequence of 4230 nucleotides contains four open reading frames (ORF) and is most closely related (59.3 %) to tobacco bushy top virus, a member of the genus Umbravirus. These data suggest that OPMV is a new umbravirus.

  15. Low numbers of repeat units in variable number of tandem repeats (VNTR) regions of white spot syndrome virus are correlated with disease outbreaks.

    PubMed

    Hoa, T T T; Zwart, M P; Phuong, N T; de Jong, M C M; Vlak, J M

    2012-11-01

    White spot syndrome virus (WSSV) is the most important pathogen in shrimp farming systems worldwide including the Mekong Delta, Vietnam. The genome of WSSV is characterized by the presence of two major 'indel regions' found at ORF14/15 and ORF23/24 (WSSV-Thailand) and three regions with variable number tandem repeats (VNTR) located in ORF75, ORF94 and ORF125. In the current study, we investigated whether or not the number of repeat units in the VNTRs correlates with virus outbreak status and/or shrimp farming practice. We analysed 662 WSSV samples from individual WSSV-infected Penaeus monodon shrimp from 104 ponds collected from two important shrimp farming regions of the Mekong Delta: Ca Mau and Bac Lieu. Using this large data set and statistical analysis, we found that for ORF94 and ORF125, the mean number of repeat units (RUs) in VNTRs was significantly lower in disease outbreak ponds than in non-outbreak ponds. Although a higher mean RU number was observed in the improved-extensive system than in the rice-shrimp or semi-intensive systems, these differences were not significant. VNTR sequences are thus not only useful markers for studying WSSV genotypes and populations, but specific VNTR variants also correlate with disease outbreaks in shrimp farming systems.

  16. Characterization of a molluscum contagiosum virus homolog of the vaccinia virus p37K major envelope antigen.

    PubMed

    Blake, N W; Porter, C D; Archard, L C

    1991-07-01

    We present the first nucleotide sequence data for molluscum contagiosum virus (MCV), an unclassified poxvirus. A 2,276-bp XhoI fragment from a near left-terminal fragment of MCV subtype I (MCVI) and a 1,920-bp XhoI fragment from the corresponding locus of MCV subtype II (MCVII) were sequenced and analyzed for open reading frames (ORFs). A large, complete ORF of 1,167 bp was present in both fragments. The putative polypeptide has a calculated molecular mass of 43 kDa (p43K protein) and was shown to have a high degree of homology to the vaccinia virus p37K major envelope antigen (40% amino acid identity and 22% conservative changes). The nucleotide content of the MCV fragments sequenced was 66% G or C. The codon usage within the gene for p43K reflected this high G + C content, with position 3 of codons being predominantly G or C (82 and 87% for MCVI and MCVII, respectively). The MCV p43K-encoding gene has motifs immediately upstream which are similar to those required for vaccinia virus late gene expression. The location and direction of transcription of the MCV p43K-encoding gene were equivalent to those of the vaccinia virus p37K gene, revealing similarity in genetic organization between MCV and vaccinia virus. Another, incomplete ORF was identified downstream of the p43K-encoding gene in both MCVI and MCVII. The sequence immediately upstream of this ORF overlapped the termination codon of the p43K-encoding gene and contained a motif which had homology to the derived consensus sequence for vaccinia virus early gene promoters.

  17. Cysteine Usage in Sulfolobus Spindle-Shaped Virus 1 And Extension to Hyperthermophilic Viruses in General

    SciTech Connect

    Menon, S.K.; Maaty, W.S.; Corn, G.J.; Kwok, S.C.; Eilers, B.J.; Kraft, P.; Gillitzer, E.; Young, M.J.; Bothner, B.; Lawrence, C.M.

    2009-05-26

    Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs worldwide. The type virus, Sulfolobus spindle-shaped virus 1 (SSV1), has a double-stranded DNA genome that contains 34 open reading frames (ORFs). Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, tertiary protein structure can provide insight into protein function. We have thus undertaken a systematic investigation of the SSV1 proteome and report here on the F112 gene product. Biochemical, proteomic and structural studies reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Notably, the structure contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses, and reveals decreased cysteine content in the membrane proteins of hyperthermophilic viruses infecting Sulfolobales. The evolutionary implications of the SSV1 distribution are discussed.

  18. The complete genomic sequence of pepper yellow leaf curl virus (PYLCV) and its implications for our understanding of evolution dynamics in the genus polerovirus.

    PubMed

    Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

    2013-01-01

    We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range.

  19. The Complete Genomic Sequence of Pepper Yellow Leaf Curl Virus (PYLCV) and Its Implications for Our Understanding of Evolution Dynamics in the Genus Polerovirus

    PubMed Central

    Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

    2013-01-01

    We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range. PMID:23936244

  20. The complete genomic sequence of pepper yellow leaf curl virus (PYLCV) and its implications for our understanding of evolution dynamics in the genus polerovirus.

    PubMed

    Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

    2013-01-01

    We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range. PMID:23936244

  1. Drought stress differentially regulates the expression of small open reading frames (sORFs) in Arabidopsis roots and shoots.

    PubMed

    Rasheed, Sultana; Bashir, Khurram; Nakaminami, Kentaro; Hanada, Kousuke; Matsui, Akihiro; Seki, Motoaki

    2016-08-01

    Characterizing the molecular mechanisms governing the response of plant roots and shoots to drought stress could aid the development of strategies aiming to ameliorate drought stress. Small open reading frames (sORFs), putatively encoding small peptides, may play a significant role in the response to different abiotic stresses. Microarray analyses revealed that after 5, 7 and 9 d of a drought treatment, 2, 77, and 104 sORFs were up-regulated in roots, respectively; while the number of upregulated sORFs in shoots was 12, 45, and 158, respectively. RT-qPCR analysis confirmed the up-regulated expression of ATRIKEN29196 and ATRIKEN32280 specifically in roots. The identified upregulated sORFs, particularly those in roots, may contribute to drought stress tolerance.

  2. Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Wilson, Stuart A.; Kalra, Priti; Golovanov, Alexander P.

    2014-01-01

    The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions. PMID:24550725

  3. Analysis of the complete genome of Tembusu virus, a flavivirus isolated from ducks in China.

    PubMed

    Tang, Y; Diao, Y; Gao, X; Yu, C; Chen, L; Zhang, D

    2012-08-01

    During investigations into the outbreak of duck viral infection in 2010 in China, with a severe drop in egg production, a flavivirus was isolated from the affected ducks. It was characterized as a Tembusu virus (TMUV). In this study, we obtained a complete genome sequence of Tembusu virus using RT-PCR and RACE techniques. TMUV genome is a singled-stranded RNA, with 10,990 nucleotides in length, and contains a single open reading frame (3410 amino acids) encoding 11 viral proteins with 5'and 3'non-translated regions (NTRs) of 142 and 618 nt, respectively. We characterized the open reading frame (ORF) with respect to gene sizes, cleavage sites and potential glycosylation sites. The different genomic regions of the virus were also compared with those of six other flaviviruses including Japanese encephalitis virus, West Nile virus (WNV), dengue-2 virus, yellow fever virus, tick-borne encephalitis virus (TBEV) and Bagaza virus. TMUV demonstrated the highest similarity to Bagaza virus. The result of entire ORF scanning shows that TMUV was close to Bagaza viruses in genetic relatedness. These data demonstrate that TMUV is a unique virus among the mosquito-borne flaviviruses and also provide a useful reference for a critically important study to determine why TMUV is a serious pathogen for ducks.

  4. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.

    PubMed

    Kuno, G; Chang, G-J J

    2007-01-01

    Many members of the genus Flavivirus are the agents of important diseases of humans, livestock, and wildlife. Currently, no complete genome sequence is available for the three African viruses, Bagaza, Zika, and Kedougou viruses, each representing a distinct virus subgroup according to the latest virus classification. In this study, we obtained a complete genome sequence of each of those three viruses and characterized the open reading frames (ORFs) with respect to gene sizes, cleavage sites, potential glycosylation sites, distribution of cysteine residues, and unique motifs. The sequences of the three viruses were then scanned across the entire length of the ORF against available sequences of other African flaviviruses and selected reference viruses for genetic relatedness. The data collectively indicated that Kedougou virus was close to dengue viruses but nonetheless distinct, while Bagaza virus shared genetic relatedness with West Nile virus in several genomic regions. In the non-coding regions, it was found that a particular organizational pattern of conserved sequences in the 3' terminal region generally correlated with the current virus grouping.

  5. Identification of essential and non-essential genes in Ambystoma tigrinum virus.

    PubMed

    Aron, Mariah M; Allen, Alexander G; Kromer, Mathew; Galvez, Hector; Vigil, Brianna; Jancovich, James K

    2016-06-01

    Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155kbp in length and they are predicted to encode around 90-125 genes. Currently, our knowledge of the function of ∼50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV.

  6. Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

    PubMed Central

    Schalk, H J; Matzeit, V; Schiller, B; Schell, J; Gronenborn, B

    1989-01-01

    By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses. Images PMID:2721484

  7. Identification of essential and non-essential genes in Ambystoma tigrinum virus.

    PubMed

    Aron, Mariah M; Allen, Alexander G; Kromer, Mathew; Galvez, Hector; Vigil, Brianna; Jancovich, James K

    2016-06-01

    Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155kbp in length and they are predicted to encode around 90-125 genes. Currently, our knowledge of the function of ∼50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV. PMID:27025572

  8. A new positive-strand RNA virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta.

    PubMed

    Valles, Steven M; Strong, Charles A; Hashimoto, Yoshifumi

    2007-09-01

    We report the discovery of a new virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. This virus represents the second identified from this ant species. It is provisionally named Solenopsis invicta virus 2 (SINV-2). The SINV-2 genome was constructed by compiling sequences from successive 5' RACE reactions, a 3' RACE reaction, and expressed sequence tag, c246 (accession number EH413675), from a fire ant expression library. The SINV-2 genome structure was monopartite, polycistronic and RNA-based. The genome consensus sequence (EF428566) was 11,303 nucleotides in length, excluding the poly(A) tail present on the 3' end. Analysis of the genome revealed 4 major open reading frames (ORFs; comprised of > or =100 codons) and 5 minor ORFs (comprised of 50-99 codons) in the sense orientation. No large ORFs were found in the inverse orientation suggesting that the SINV-2 genome was from a positive-strand RNA virus. Further evidence for this conclusion includes abolished RT-PCR amplification by RNase treatment of SINV-2 nucleic acid template, and failure to amplify without first conducting cDNA synthesis. Blastp analysis indicated that ORF 4 contained conserved domains of an RNA-dependent RNA polymerase, helicase, and protease, characteristic of positive-strand RNA viruses. However, the protease domain and putative structural proteins (ORFs 1, 2, and 3) were less well conserved. Phylogenetic analysis of the RdRp, helicase, and ORF 1 indicate unique placement of SINV-2 exclusive from the Dicistroviridae, iflaviruses, Picornaviridae, and plant small RNA viruses. PMID:17477949

  9. C1orf163/RESA1 is a novel mitochondrial intermembrane space protein connected to respiratory chain assembly.

    PubMed

    Kozjak-Pavlovic, Vera; Prell, Florian; Thiede, Bernd; Götz, Monika; Wosiek, Dominik; Ott, Christine; Rudel, Thomas

    2014-02-20

    Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60kDa and 150kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).

  10. Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition.

    PubMed

    Mori, Kohji; Nihei, Yoshihiro; Arzberger, Thomas; Zhou, Qihui; Mackenzie, Ian R; Hermann, Andreas; Hanisch, Frank; Kamp, Frits; Nuscher, Brigitte; Orozco, Denise; Edbauer, Dieter; Haass, Christian

    2016-09-01

    Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci. PMID:27461252

  11. C11orf83, a Mitochondrial Cardiolipin-Binding Protein Involved in bc1 Complex Assembly and Supercomplex Stabilization

    PubMed Central

    Foti, Michelangelo; Raemy, Etienne; Vaz, Frédéric Maxime; Martinou, Jean-Claude; Bairoch, Amos

    2015-01-01

    Mammalian mitochondria may contain up to 1,500 different proteins, and many of them have neither been confidently identified nor characterized. In this study, we demonstrated that C11orf83, which was lacking experimental characterization, is a mitochondrial inner membrane protein facing the intermembrane space. This protein is specifically associated with the bc1 complex of the electron transport chain and involved in the early stages of its assembly by stabilizing the bc1 core complex. C11orf83 displays some overlapping functions with Cbp4p, a yeast bc1 complex assembly factor. Therefore, we suggest that C11orf83, now called UQCC3, is the functional human equivalent of Cbp4p. In addition, C11orf83 depletion in HeLa cells caused abnormal crista morphology, higher sensitivity to apoptosis, a decreased ATP level due to impaired respiration and subtle, but significant, changes in cardiolipin composition. We showed that C11orf83 binds to cardiolipin by its α-helices 2 and 3 and is involved in the stabilization of bc1 complex-containing supercomplexes, especially the III2/IV supercomplex. We also demonstrated that the OMA1 metalloprotease cleaves C11orf83 in response to mitochondrial depolarization, suggesting a role in the selection of cells with damaged mitochondria for their subsequent elimination by apoptosis, as previously described for OPA1. PMID:25605331

  12. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    PubMed Central

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J; Weston, Ria; Williams, Jason G; Rossi, Marianna N; Galehdari, Hamid; Krahn, Juno; Wan, Alexander; Trembath, Richard C; Crosby, Andrew H; Ahel, Dragana; Hay, Ron; Ladurner, Andreas G; Timinszky, Gyula; Williams, R Scott; Ahel, Ivan

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. PMID:23481255

  13. C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human

    PubMed Central

    Bontems, Franck; Fish, Richard J.; Borlat, Irene; Lembo, Frédérique; Chocu, Sophie; Chalmel, Frédéric; Borg, Jean-Paul; Pineau, Charles; Neerman-Arbez, Marguerite; Bairoch, Amos; Lane, Lydie

    2014-01-01

    Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) – like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis. PMID:24475127

  14. Functional similarities between phage lambda Orf and Escherichia coli RecFOR in initiation of genetic exchange.

    PubMed

    Maxwell, Karen L; Reed, Patricia; Zhang, Rong-Guang; Beasley, Steven; Walmsley, Adrian R; Curtis, Fiona A; Joachimiak, Andrej; Edwards, Aled M; Sharples, Gary J

    2005-08-01

    Genetic recombination in bacteriophage lambda relies on DNA end processing by Exo to expose 3'-tailed strands for annealing and exchange by beta protein. Phage lambda encodes an additional recombinase, Orf, which participates in the early stages of recombination by supplying a function equivalent to the Escherichia coli RecFOR complex. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with ssDNA-binding protein. In this study, we purified the Orf protein, analyzed its biochemical properties, and determined its crystal structure at 2.5 angstroms. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3'-tailed, or 5'-tailed substrates. An interaction between Orf and ssDNA-binding protein was indicated by far Western analysis. The functional similarities between Orf and RecFOR are discussed in relation to the early steps of recombinational exchange and the interplay between phage and bacterial recombinases. PMID:16076958

  15. C9orf72’s Interaction with Rab GTPases—Modulation of Membrane Traffic and Autophagy

    PubMed Central

    Tang, Bor L.

    2016-01-01

    Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 (C9orf72) is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF) complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1) autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings. PMID:27774051

  16. Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality.

    PubMed

    Park, Arnold; Yun, Tatyana; Hill, Terence E; Ikegami, Tetsuro; Juelich, Terry L; Smith, Jennifer K; Zhang, Lihong; Freiberg, Alexander N; Lee, Benhur

    2016-04-01

    Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design. PMID:26781134

  17. Positive expression of protein chromosome 9 open reading frame 86 (C9orf86) correlated with poor prognosis in non-small cell lung cancer patients

    PubMed Central

    Peng, Gui-Lin; Tao, Ya-Lan; Wu, Qi-Nian; Zhang, Yu

    2016-01-01

    Background Chromosome 9 open reading frame 86 (C9orf86) is a novel subfamily of GTPases. Previous studies have implicated C9orf86 as a potential oncogene. Methods C9orf86 expression was detected in non-small cell lung cancer (NSCLC) cell lines and human bronchial epithelial (16HBE) cell lines by RT-PCR and western blotting. Immunohistochemistry (IHC) was used to detect 180 consecutive NSCLC specimens and 16 normal lung tissues. The correlation between C9orf86 expression and clinicopathological parameters was evaluated. Kaplan-Meier survival analysis and Cox hazards ratio models were used to estimate the effect of C9orf86 expression on survival. Results C9orf86 was expressed in the cytoplasm in 74 of 180 (41.11%) NSCLC specimens. In clinical pathology analysis, C9orf86 expression significantly correlated with lymph node metastasis and clinical stage significantly (P<0.05). Multivariable analysis confirmed that C9orf86 expression increased the risk of death after adjusting for other clinicopathological factors (P<0.01). Overall survival (OS) and disease-free survival (DFS) were significantly prolonged in the C9orf86 negative group compared to the C9orf86 positive group (P<0.001). Adjuvant chemotherapy prolonged OS and DFS in resected NSCLC patients with C9orf86 negative expression (P<0.001) but not C9orf86 positive. Conclusions Positive expression of C9orf86 is an independent prognostic factor for NSCLC patients, and C9orf86 may serve as a prognostic biomarker for patients with NSCLC. PMID:27499931

  18. The Hepatitis E virus intraviral interactome

    PubMed Central

    Osterman, Andreas; Stellberger, Thorsten; Gebhardt, Anna; Kurz, Marisa; Friedel, Caroline C.; Uetz, Peter; Nitschko, Hans; Baiker, Armin; Vizoso-Pinto, Maria G.

    2015-01-01

    Hepatitis E virus (HEV) is an emerging virus causing epidemic acute hepatitis in developing countries as well as sporadic cases in industrialized countries. The life cycle of HEV is still poorly understood and the lack of efficient cell culture systems and animal models are the principal limitations for a detailed study of the viral replication cycle. Here we exhaustively examine all possible intraviral protein-protein interactions (PPIs) of HEV by systematic Yeast two-hybrid (Y2H) and LuMPIS screens, providing a basis for studying the function of these proteins in the viral replication cycle. Key PPIs correlate with the already published HEV 3D structure. Furthermore, we report 20 novel PPIs including the homodimerization of the RNA dependent RNA polymerase (RdRp), the self-interaction of the papain like protease, and ORF3 interactions with the papain-like protease and putative replicase components: RdRp, methylase and helicase. Furthermore, we determined the dissociation constant (Kd) of ORF3 interactions with the viral helicase, papain-like protease and methylase, which suggest a regulatory function for ORF3 in orchestrating the formation of the replicase complex. These interactions may represent new targets for antiviral drugs. PMID:26463011

  19. White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species.

    PubMed

    Sindhupriya, M; Saravanan, P; Otta, S K; Amarnath, C Bala; Arulraj, R; Bhuvaneswari, T; Praveena, P Ezhil; Jithendran, K P; Ponniah, A G

    2014-08-21

    White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

  20. Construction and expression of a heterologous protein in Lactococcus lactis by using the nisin-controlled gene expression system: the case of the PRRSV ORF6 gene.

    PubMed

    Wang, Z H; Wang, Y L; Zeng, X Y

    2014-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, exerting significant economic effects on the swine industry worldwide. However, none of the current commercially available vaccines can completely prevent respiratory infection, trans-placental transmission, pig-to-pig transmission of the virus, or maintain immune protection in sows. This study provides information on PRRSV and a review of available options for PRRS control strategies based on its pathogenic characteristics, immune properties, and biological characteristics. In this study, the nisin-controlled expression system of Lactococcus lactis was selected as a vector to express the ORF6 gene of PRRSV. Food-grade recombinant, L. lactis PNZ8149/NZ3900-M/PRRS, which contained the lactose operon, was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 19 kDa. Furthermore, the recombinant protein was located on the surface of L. lactis and showed reactogenicity with the antibody against PRRSV. Results of this study are expected to lay a theoretical foundation for development of genetically engineered L. lactis mucosal vaccines and to provide information related to its immune activity and adjuvant effects. PMID:24634130

  1. The genome organization of banana bunchy top virus: analysis of six ssDNA components.

    PubMed

    Burns, T M; Harding, R M; Dale, J L

    1995-06-01

    We have cloned, sequenced and analysed an additional five circular ssDNA components of banana bunchy top virus (BBTV) which we have called components 2, 3, 4, 5 and 6. These components were present in all BBTV infections tested. Four of these components (components 3, 4, 5 and 6) had one large open reading frame (ORF) in the virion sense located 3' of a stem-loop structure. Each ORF had a potential TATA box and one or two potential polyadenylation signals associated with it and each polyadenylation signal had an associated GC-rich region containing the trinucleotide sequence TTG. A number of ORFs were identified in component 2 but none of these had appropriately located potential TATA boxes and polyadenylation signals associated with them. None of the ORF amino acid sequences nor the full DNA sequences of any of the components had significant sequence identity with any known protein or nucleic acid sequences. However, the ORF of component 4 encoded a 30 residue hydrophobic domain which may indicate that this ORF encoded a transmembrane protein. Further, the ORFs of components 3 and 5 potentially encoded proteins of about 20 kDa, the size of the BBTV coat protein. There were two regions of sequence identity between the five components described here and the previously described component 1. Each component contained a conserved stem-loop structure and a nonanucleotide potential TATA box which was 5' of the large virion-sense ORF in five of the components. The stem-loop structures were incorporated in a common region (CR-SL) of 69 nucleotides which was 62% identical between components. All six BBTV components also contained a major common region (CR-M) which was located 5' of the CR-SL in each component, in the non-coding region and was 76% identical over 92 nucleotides. Each CR-M contained a near-complete 16 nucleotide direct repeat and a GC-box which was similar to the rightward promoter element found in wheat dwarf geminivirus. From these results, BBTV appears to

  2. Varicella-Zoster Virus Open Reading Frame 66 Protein Kinase and Its Relationship to Alphaherpesvirus US3 Kinases

    PubMed Central

    Erazo, Angela

    2014-01-01

    The varicella-zoster virus (VZV) open reading frame (ORF) 66 encodes a basophilic kinase orthologous to the US3 protein kinases found in all alphaherpesviruses. This review summarizes current information on the ORF66 kinase, and outlines apparent differences from other US3 kinases, as well as some of the conserved functions. One critical difference is the VZV ORF66 kinase targeting of the major regulatory VZV IE62 protein to control its nuclear import and assembly into the VZV virion, which is so far unprecedented in the alphaherpesviruses. However, ORF66 targets some cellular targets which are also targeted by US3 kinases of other herpesviruses, including the histone deacetylase-1 and 2 proteins, pathways that lead to changes in actin dynamics, and the targeting of substrates of protein kinase A, including the nuclear matrix protein matrin 3. PMID:20186610

  3. Identification of chicken enterovirus-like viruses, duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses.

    PubMed

    Todd, D; Smyth, V J; Ball, N W; Donnelly, B M; Wylie, M; Knowles, N J; Adair, B M

    2009-02-01

    Earlier work identified and biologically characterized antigenically distinct enterovirus-like viruses (ELVs) of chickens. Three of these ELVs can now be identified as astroviruses. Characterization involved the use of a hitherto undescribed, degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) to amplify astrovirus open reading frame (ORF) 1b-specific cDNA fragments followed by nucleotide sequence determination and analysis of the amplified fragments. ELV-1 was confirmed as an isolate of the astrovirus avian nephritis virus (ANV). ELV-4 (isolate 612) and ELV-3 (isolates FP3 and 11672) were antigenically and genetically related to the second characterized astrovirus of chickens, namely chicken astrovirus (CAstV). Using indirect immunofluorescence, the FP3 and 11672 ELV-3 isolates were very closely related to one another, and less closely related to ELV-4 and the previously described CAstV (P22 18.8.00 reference isolate). Comparative analyses based on the ORF 1b amplicon sequences showed that the FP3 and 11672 ELV-3 isolates shared high nucleotide (95%) and amino acid (98%) identities with one another, and lower nucleotide (76% to 79%) and amino acid (84% to 85%) identity levels with ELV-4 and the reference CAstV P22 18.8.00 isolates. The combined degenerate primer RT-PCR and sequencing methods also provided a nucleotide sequence specific to duck hepatitis virus type 2 (DHV-2) (renamed duck astrovirus) and duck hepatitis virus type 3 (DHV-3), which, for the first time, can also be identified as an astrovirus. Phylogenetic analyses based on the amplified ORF 1b sequences showed that ANV was the most distantly related avian astrovirus, with DHV-3 being more closely related to turkey astrovirus type 2 than DHV-2.

  4. Similarity-based gene detection: using COGs to find evolutionarily-conserved ORFs

    PubMed Central

    Powell, Bradford C; Hutchison, Clyde A

    2006-01-01

    Background Experimental verification of gene products has not kept pace with the rapid growth of microbial sequence information. However, existing annotations of gene locations contain sufficient information to screen for probable errors. Furthermore, comparisons among genomes become more informative as more genomes are examined. We studied all open reading frames (ORFs) of at least 30 codons from the genomes of 27 sequenced bacterial strains. We grouped the potential peptide sequences encoded from the ORFs by forming Clusters of Orthologous Groups (COGs). We used this grouping in order to find homologous relationships that would not be distinguishable from noise when using simple BLAST searches. Although COG analysis was initially developed to group annotated genes, we applied it to the task of grouping anonymous DNA sequences that may encode proteins. Results "Mixed COGs" of ORFs (clusters in which some sequences correspond to annotated genes and some do not) are attractive targets when seeking errors of gene predicion. Examination of mixed COGs reveals some situations in which genes appear to have been missed in current annotations and a smaller number of regions that appear to have been annotated as gene loci erroneously. This technique can also be used to detect potential pseudogenes or sequencing errors. Our method uses an adjustable parameter for degree of conservation among the studied genomes (stringency). We detail results for one level of stringency at which we found 83 potential genes which had not previously been identified, 60 potential pseudogenes, and 7 sequences with existing gene annotations that are probably incorrect. Conclusion Systematic study of sequence conservation offers a way to improve existing annotations by identifying potentially homologous regions where the annotation of the presence or absence of a gene is inconsistent among genomes. PMID:16423288

  5. Jump from pre-mutation to pathologic expansion in C9orf72.

    PubMed

    Xi, Zhengrui; van Blitterswijk, Marka; Zhang, Ming; McGoldrick, Philip; McLean, Jesse R; Yunusova, Yana; Knock, Erin; Moreno, Danielle; Sato, Christine; McKeever, Paul M; Schneider, Raphael; Keith, Julia; Petrescu, Nicolae; Fraser, Paul; Tartaglia, Maria Carmela; Baker, Matthew C; Graff-Radford, Neill R; Boylan, Kevin B; Dickson, Dennis W; Mackenzie, Ian R; Rademakers, Rosa; Robertson, Janice; Zinman, Lorne; Rogaeva, Ekaterina

    2015-06-01

    An expanded G4C2 repeat in C9orf72 represents the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the lower limit for pathological expansions is unknown (the suggested cutoff is 30 repeats). It has been proposed that the expansion might have occurred only once in human history and subsequently spread throughout the population. However, our present findings support a hypothesis of multiple origins for the expansion. We report a British-Canadian family in whom a ∼70-repeat allele from the father (unaffected by ALS or FTLD at age 89 years) expanded during parent-offspring transmission and started the first generation affected by ALS (four children carry an ∼1,750-repeat allele). Epigenetic and RNA-expression analyses further discriminated the offspring's large expansions (which were methylated and associated with reduced C9orf72 expression) from the ∼70-repeat allele (which was unmethylated and associated with upregulation of C9orf72). Moreover, RNA foci were only detected in fibroblasts from offspring with large expansions, but not in the father, who has the ∼70-repeat allele. All family members with expansions were found to have an ancient known risk haplotype, although it was inherited on a unique 5-Mb genetic backbone. We conclude that small expansions (e.g., 70 repeats) might be considered "pre-mutations" to reflect their propensity to expand in the next generation. Follow-up studies might help explain the high frequency of ALS- or FTLD-affected individuals with an expansion but without a familial history (e.g., 21% among Finnish ALS subjects). PMID:26004200

  6. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    SciTech Connect

    Lebrun, Marielle; Thelen, Nicolas; Thiry, Marc; Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia; Di Valentin, Emmanuel; Bontems, Sébastien; Sadzot-Delvaux, Catherine

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  7. Semi-automated quantification of C9orf72 expansion size reveals inverse correlation between hexanucleotide repeat number and disease duration in frontotemporal degeneration.

    PubMed

    Suh, EunRan; Lee, Edward B; Neal, Donald; Wood, Elisabeth M; Toledo, Jon B; Rennert, Lior; Irwin, David J; McMillan, Corey T; Krock, Bryan; Elman, Lauren B; McCluskey, Leo F; Grossman, Murray; Xie, Sharon X; Trojanowski, John Q; Van Deerlin, Vivianna M

    2015-09-01

    We investigated whether chromosome 9 open reading frame 72 hexanucleotide repeat expansion (C9orf72 expansion) size in peripheral DNA was associated with clinical differences in frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) linked to C9orf72 repeat expansion mutations. A novel quantification workflow was developed to measure C9orf72 expansion size by Southern blot densitometry in a cross-sectional cohort of C9orf72 expansion carriers with FTD (n = 39), ALS (n = 33), both (n = 35), or who are unaffected (n = 21). Multivariate linear regressions were performed to assess whether C9orf72 expansion size from peripheral DNA was associated with clinical phenotype, age of disease onset, disease duration and age at death. Mode values of C9orf72 expansion size were significantly shorter in FTD compared to ALS (p = 0.0001) but were not associated with age at onset in either FTD or ALS. A multivariate regression model correcting for patient's age at DNA collection and disease phenotype revealed that C9orf72 expansion size is significantly associated with shorter disease duration (p = 0.0107) for individuals with FTD, but not with ALS. Despite considerable somatic instability of the C9orf72 expansion, semi-automated expansion size measurements demonstrated an inverse relationship between C9orf72 expansion size and disease duration in patients with FTD. Our finding suggests that C9orf72 repeat size may be a molecular disease modifier in FTD linked to hexanucleotide repeat expansion.

  8. Proteolytic processing of Porcine Reproductive and Respiratory Syndrome Virus nsp2 replicase protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One critical step in porcine reproductive and respiratory syndrome virus (PRRSV) replication is the proteolytic processing of the ORF1 polyprotein (replicase). The replicase polyprotein is generally believed to be processed to generate at least 12 smaller nonstructural proteins (nsps) involved in r...

  9. The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

    PubMed

    de Munnik, Sabrina M; van der Lee, Rosan; Velders, Daniëlle M; van Offenbeek, Jody; Smits-de Vries, Laura; Leurs, Rob; Smit, Martine J; Vischer, Henry F

    2016-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74. PMID:26931381

  10. Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies*

    PubMed Central

    Patsalo, Vadim; Yondola, Mark A.; Luan, Bowu; Shoshani, Ilana; Kisker, Caroline; Green, David F.; Raleigh, Daniel P.; Hearing, Patrick

    2012-01-01

    The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity. PMID:22573317

  11. Gene repertoire of amoeba-associated giant viruses.

    PubMed

    Colson, Philippe; Raoult, Didier

    2010-01-01

    Acanthamoeba polyphaga mimivirus, Marseillevirus, and Sputnik, a virophage, are intra-amoebal viruses that have been isolated from water collected in cooling towers. They have provided fascinating data and have raised exciting questions about viruses definition and evolution. Mimivirus and Marseillevirus have been classified in the nucleo-cytoplasmic large DNA viruses (NCLDVs) class. Their genomes are the largest and fifth largest viral genomes sequenced so far. The gene repertoire of these amoeba-associated viruses can be divided into four groups: the core genome, genes acquired by lateral gene transfer, duplicated genes, and ORFans. Open reading frames (ORFs) that have homologs in the NCLDVs core gene set represent 2.9 and 6.1% of the Mimivirus and Marseillevirus gene contents, respectively. A substantial proportion of the Mimivirus, Marseillevirus and Sputnik ORFs exhibit sequence similarities to homologs found in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes might have resulted from acquisitions by lateral gene transfers, implicating sympatric bacteria and viruses with an intra-amoebal lifestyle. In addition, lineage-specific gene expansion may have played a major role in the genome shaping. Altogether, the data so far accumulated on amoeba-associated giant viruses are a powerful incentive to isolate and study additional strains to gain better understanding of their pangenome. PMID:20551685

  12. Attenuation of the adaptive immune response in rhesus macaques infected with simian varicella virus lacking open reading frame 61.

    PubMed

    Meyer, Christine; Kerns, Amelia; Haberthur, Kristen; Dewane, Jesse; Walker, Joshua; Gray, Wayne; Messaoudi, Ilhem

    2013-02-01

    Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Infection of rhesus macaques (RM) with the homologous simian varicella virus (SVV) recapitulates hallmarks of VZV infection. We have shown that an antisense transcript of SVV open reading frame 61 (ORF61), a viral transactivator, was detected most frequently in latently infected RM sensory ganglia. In this study, we compared disease progression, viral replication, immune response, and the establishment of latency following intrabronchial infection with a recombinant SVV lacking ORF61 (SVVΔORF61) to those following infection with wild-type (WT) SVV. Varicella severity and viral latency within sensory ganglia were comparable in RMs infected with SVVΔORF61 and WT SVV. In contrast, viral loads, B and T cell responses, and plasma inflammatory cytokine levels were decreased in RMs infected with SVVΔORF61. To investigate the mechanisms underlying the reduced adaptive immune response, we compared acute SVV gene expression, frequency and proliferation of dendritic cell (DC) subsets, and the expression of innate antiviral genes in bronchoalveolar lavage (BAL) samples. The abundance of SVV transcripts in all kinetic classes was significantly decreased in RMs infected with SVVΔORF61. In addition, we detected a higher frequency and proliferation of plasmacytoid dendritic cells in BAL fluid at 3 days postinfection in RMs infected with SVVΔORF61, which was accompanied by a slight increase in type I interferon gene expression. Taken together, our data suggest that ORF61 plays an important role in orchestrating viral gene expression in vivo and interferes with the host antiviral interferon response.

  13. Construction and immunogenicity of a recombinant pseudorabies virus co-expressing porcine circovirus type 2 capsid protein and interleukin 18.

    PubMed

    Zheng, Lan-lan; Guo, Xiao-qing; Zhu, Qian-lei; Chao, An-jun; Fu, Peng-fei; Wei, Zhan-yong; Wang, Shu-juan; Chen, Hong-ying; Cui, Bao-an

    2015-04-01

    A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice (n=10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3(+), CD4(+) and CD8(+) T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs.

  14. Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies.

    PubMed Central

    Wirblich, C; Thiel, H J; Meyers, G

    1996-01-01

    The 7.5-kb plus-stranded genomic RNA of rabbit hemorrhagic disease virus contains two open reading frames of 7 kb (ORF1) and 351 nucleotides (ORF2) that cover nearly 99% of the genome. The aim of the present study was to identify the proteins encoded in these open reading frames. To this end, a panel of region-specific antisera was generated by immunization of rabbits with bacterially expressed fusion proteins that encompass in total 95% of the ORF1 polyprotein and almost the complete ORF2 polypeptide. The antisera were used to analyze the in vitro translation products of purified virion RNA of rabbit hemorrhagic disease virus. Our studies show that the N-terminal half of the ORF1 polyprotein is proteolytically cleaved to yield three nonstructural proteins of 16, 23, and 37 kDa (p16, p23, and p37, respectively). In addition, a cleavage product of 41 kDa which is composed of VPg and a putative nonstructural protein of approximately 30 kDa was identified. Together with the results of previous studies which identified a trypsin-like cysteine protease (TCP) of 15 kDa, a putative RNA polymerase (pol) of 58 kDa, and the major capsid protein VP60, our data establish the following gene order in ORF1: NH2-p16-p23-p37 (helicase)-p30-VPg-TCP-pol-VP60-COOH. Immunoblot analyses showed that a minor structural protein of 10 kDa is encoded in ORF2. The data provide the first complete genetic map of a calicivirus. The map reveals a remarkable similarity between caliciviruses and picornaviruses with regard to the number and order of the genes that encode the nonstructural proteins. PMID:8892921

  15. Complete genome sequencing of a genotype 3 hepatitis E virus strain identified in a swine farm in Italy.

    PubMed

    Di Bartolo, Ilaria; Angeloni, Giorgia; Monini, Marina; Maione, Ester; Marrone, Raffaele; Ostanello, Fabio; Ruggeri, Franco Maria

    2016-01-01

    In this study, we investigated hepatitis E virus (HEV) infection in piglets sampled in two farms in southern Italy. The virus was detected in 11 out of 15 animals tested. Based on sequence analysis, the 6 Italian strains examined belonged to two clusters containing both swine and human strains of either genotype 3 subtype e or f from Europe and Japan. The two Italian strain clusters shared nucleotide identity of 81.8% and 87.5% in the ORF2 (capsid protein) and ORF1 (RdRp) diagnostic fragments, respectively, confirming the heterogeneity of genotype 3 viruses circulating in pigs in Italy. The complete genome of one genotype 3 subtype e strain and the full ORF2 and ORF3 coding regions of one of the genotype 3f strains, obtained in this study, were compared to other HEV sequences available on line (NCBI database). The results of analysis showed that porcine strains clustered together with human and swine strains detected in Europe. Most changes in the coding region corresponded to synonymous mutations, and only the ORF3 showed a positive selection. Further, analyses are needed to understand the clinical significance of HEV genotypes and subtypes.

  16. ATNX2 is not a regulatory gene in Italian amyotrophic lateral sclerosis patients with C9ORF72 GGGGCC expansion.

    PubMed

    Chiò, Adriano; Mora, Gabriele; Sabatelli, Mario; Caponnetto, Claudia; Lunetta, Christian; Traynor, Bryan J; Johnson, Janel O; Nalls, Mike A; Calvo, Andrea; Moglia, Cristina; Borghero, Giuseppe; Trojsi, Francesca; La Bella, Vincenzo; Volanti, Paolo; Simone, Isabella; Salvi, Fabrizio; Logullo, Francesco O; Riva, Nilo; Carrera, Paola; Giannini, Fabio; Mandrioli, Jessica; Tanel, Raffaella; Capasso, Margherita; Tremolizzo, Lucio; Battistini, Stefania; Murru, Maria Rita; Origone, Paola; Zollino, Marcella; Penco, Silvana; Mazzini, Letizia; D'Alfonso, Sandra; Restagno, Gabriella; Brunetti, Maura; Barberis, Marco; Conforti, Francesca L

    2016-03-01

    There are indications that both familial amyotrophic lateral sclerosis (ALS) and sporadic ALS phenotype and prognosis are partly regulated by genetic and environmental factors, supporting the theory that ALS is a multifactorial disease. The aim of this article was to assess the role of ATXN2 intermediate length repeats in a large series of Italian and Sardinian ALS patients and controls carrying a pathogenetic C9ORF72 GGGGCC hexanucleotide repeat. A total of 1972 ALS cases were identified through the database of the Italian ALS Genetic consortium, a collaborative effort including 18 ALS centers throughout Italy. The study population included: (1) 276 Italian and 57 Sardinian ALS cases who carried the C9ORF72 expansion; (2) 1340 Italian and 299 Sardinian ALS cases not carrying the C9ORF72 expansion. A total of healthy 1043 controls were also assessed. Most Italian and Sardinian cases and controls were homozygous for 22/22 or 23/23 repeats or heterozygous for 22/23 repeats of the ATXN2 gene. ATXN2 intermediate length repeats alleles (≥28) were detected in 3 (0.6%) Italian ALS cases carrying the C9ORF72 expansion, in none of the Sardinian ALS cases carrying the expansion, in 60 (4.3%) Italian cases not carrying the expansion, and in 6 (2.0%) Sardinian ALS cases without C9ORF72 expansion. Intermediate length repeat alleles were found in 12 (1.5%) Italian controls and 1 (0.84%) Sardinian controls. Therefore, ALS patients with C9ORF72 expansion showed a lower frequency of ATXN2 polyQ intermediate length repeats than both controls (Italian cases, p = 0.137; Sardinian cases, p = 0.0001) and ALS patients without C9ORF72 expansion (Italian cases, p = 0.005; Sardinian cases, p = 0.178). In our large study on Italian and Sardinian ALS patients with C9ORF72 GGGGCC hexanucleotide repeat expansion, compared to age-, gender- and ethnic-matched controls, ATXN2 polyQ intermediate length does not represent a modifier of ALS risk, differently from non-C9ORF72 mutated patients.

  17. Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone.

    PubMed

    Jengarn, Juggragarn; Wongthida, Phonphimon; Wanasen, Nanchaya; Frantz, Phanramphoei Namprachan; Wanitchang, Asawin; Jongkaewwattana, Anan

    2015-08-01

    Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDVAVCT12. Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDVAVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDVAVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate. PMID:25979733

  18. Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.

    PubMed Central

    Collins, P L; Hill, M G; Cristina, J; Grosfeld, H

    1996-01-01

    RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions. Images Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:8552680

  19. ESRRA-C11orf20 is a recurrent gene fusion in serous ovarian carcinoma.

    PubMed

    Salzman, Julia; Marinelli, Robert J; Wang, Peter L; Green, Ann E; Nielsen, Julie S; Nelson, Brad H; Drescher, Charles W; Brown, Patrick O

    2011-09-01

    Every year, ovarian cancer kills approximately 14,000 women in the United States and more than 140,000 women worldwide. Most of these deaths are caused by tumors of the serous histological type, which is rarely diagnosed before it has disseminated. By deep paired-end sequencing of mRNA from serous ovarian cancers, followed by deep sequencing of the corresponding genomic region, we identified a recurrent fusion transcript. The fusion transcript joins the 5' exons of ESRRA, encoding a ligand-independent member of the nuclear-hormone receptor superfamily, to the 3' exons of C11orf20, a conserved but uncharacterized gene located immediately upstream of ESRRA in the reference genome. To estimate the prevalence of the fusion, we tested 67 cases of serous ovarian cancer by RT-PCR and sequencing and confirmed its presence in 10 of these. Targeted resequencing of the corresponding genomic region from two fusion-positive tumor samples identified a nearly clonal chromosomal rearrangement positioning ESRRA upstream of C11orf20 in one tumor, and evidence of local copy number variation in the ESRRA locus in the second tumor. We hypothesize that the recurrent novel fusion transcript may play a role in pathogenesis of a substantial fraction of serous ovarian cancers and could provide a molecular marker for detection of the cancer. Gene fusions involving adjacent or nearby genes can readily escape detection but may play important roles in the development and progression of cancer.

  20. Transcriptome profiling of the cysticercus stage of the laboratory model Taenia crassiceps, strain ORF.

    PubMed

    García-Montoya, Gisela M; Mesa-Arango, Jairo A; Isaza-Agudelo, Juan P; Agudelo-Lopez, Sonia P; Cabarcas, Felipe; Barrera, Luis F; Alzate, Juan F

    2016-02-01

    Neurocysticercosis (NC) is a serious public health problem mainly in developing countries. NC caused by the cysticercus stage from cestode Taenia solium is considered by the WHO and ITFDE as a potentially eradicable disease. Definitive diagnosis of NC is challenging because of the unspecific clinical manifestations such as the non-definitive evidence presented by neuroimaging (in most cases) and the lack of definitive serological test. Taenia crassiceps (ORF strain) is a cestode closely related to T. solium and it has frequently been used as a source of antigens for immunodiagnostics. A murine model to study host immune response to infection has also been established by using T. crassiceps. Despite the extensive use of T. crassiceps for research, molecular information for this cestode is scarce in public databases. With the aim of providing more extensive information on T. crassiceps biology, an RNA-seq experiment and subsequent bioinformatic transcriptome processing of this cestode parasite mRNA in its cysticercus stage were carried out. A total of 227,082 read/ESTs were sequenced using the 454-GS FLX Titanium technology and assembled into 10,787 contigs. This transcriptome dataset represents new and valuable molecular information of the cestode T. crassiceps (ORF). This information will substantially improve public information and will help to achieve a better understanding of the biology of T. crassiceps and to identify target proteins for serodiagnosis and vaccination. PMID:26571070

  1. Searching for Grendel: origin and global spread of the C9ORF72 repeat expansion

    PubMed Central

    Pliner, Hannah A.; Mann, David M.; Traynor, Bryan J.

    2015-01-01

    Recent advances are uncovering more and more of the genetic architecture underlying amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative condition that affects ~6,000 Americans annually. Chief among these was the discovery that a large repeat expansion in the C9ORF72 gene is responsible for an unprecedented portion of familial and sporadic ALS cases. Much has been published on how this expansion disrupts neuronal homeostasis and how gene-based therapy might be an effective treatment in the future. Nevertheless, it is instructive to look back at the origins of this important mutation. In this opinion piece, we attempt to answer three key questions concerning C9ORF72. First, how many times did the expansion occur throughout human history? Second, how old is the expansion? And finally and perhaps most importantly, how did the expansion spread throughout Europe? We speculate that the expansion occurred only once in the past, that this event took place in the Finnish population and that the Vikings and their descendants were responsible for disseminating this mutation throughout the rest of the continent. PMID:24496499

  2. Searching for Grendel: origin and global spread of the C9ORF72 repeat expansion.

    PubMed

    Pliner, Hannah A; Mann, David M; Traynor, Bryan J

    2014-03-01

    Recent advances are uncovering more and more of the genetic architecture underlying amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative condition that affects ~6,000 Americans annually. Chief among these was the discovery that a large repeat expansion in the C9ORF72 gene is responsible for an unprecedented portion of familial and sporadic ALS cases. Much has been published on how this expansion disrupts neuronal homeostasis and how gene-based therapy might be an effective treatment in the future. Nevertheless, it is instructive to look back at the origins of this important mutation. In this opinion piece, we attempt to answer three key questions concerning C9ORF72. First, how many times did the expansion occur throughout human history? Second, how old is the expansion? And finally and perhaps most importantly, how did the expansion spread throughout Europe? We speculate that the expansion occurred only once in the past, that this event took place in the Finnish population and that the Vikings and their descendants were responsible for disseminating this mutation throughout the rest of the continent.

  3. Transcriptome profiling of the cysticercus stage of the laboratory model Taenia crassiceps, strain ORF.

    PubMed

    García-Montoya, Gisela M; Mesa-Arango, Jairo A; Isaza-Agudelo, Juan P; Agudelo-Lopez, Sonia P; Cabarcas, Felipe; Barrera, Luis F; Alzate, Juan F

    2016-02-01

    Neurocysticercosis (NC) is a serious public health problem mainly in developing countries. NC caused by the cysticercus stage from cestode Taenia solium is considered by the WHO and ITFDE as a potentially eradicable disease. Definitive diagnosis of NC is challenging because of the unspecific clinical manifestations such as the non-definitive evidence presented by neuroimaging (in most cases) and the lack of definitive serological test. Taenia crassiceps (ORF strain) is a cestode closely related to T. solium and it has frequently been used as a source of antigens for immunodiagnostics. A murine model to study host immune response to infection has also been established by using T. crassiceps. Despite the extensive use of T. crassiceps for research, molecular information for this cestode is scarce in public databases. With the aim of providing more extensive information on T. crassiceps biology, an RNA-seq experiment and subsequent bioinformatic transcriptome processing of this cestode parasite mRNA in its cysticercus stage were carried out. A total of 227,082 read/ESTs were sequenced using the 454-GS FLX Titanium technology and assembled into 10,787 contigs. This transcriptome dataset represents new and valuable molecular information of the cestode T. crassiceps (ORF). This information will substantially improve public information and will help to achieve a better understanding of the biology of T. crassiceps and to identify target proteins for serodiagnosis and vaccination.

  4. Distinct C9orf72-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity

    PubMed Central

    Krans, Amy; Sawaya, Michael R.; Paulson, Henry L.; Todd, Peter K.; Barmada, Sami J.; Ivanova, Magdalena I.

    2016-01-01

    Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity. PMID:27776165

  5. Genome annotation past, present, and future: how to define an ORF at each locus.

    PubMed

    Brent, Michael R

    2005-12-01

    Driven by competition, automation, and technology, the genomics community has far exceeded its ambition to sequence the human genome by 2005. By analyzing mammalian genomes, we have shed light on the history of our DNA sequence, determined that alternatively spliced RNAs and retroposed pseudogenes are incredibly abundant, and glimpsed the apparently huge number of non-coding RNAs that play significant roles in gene regulation. Ultimately, genome science is likely to provide comprehensive catalogs of these elements. However, the methods we have been using for most of the last 10 years will not yield even one complete open reading frame (ORF) for every gene--the first plateau on the long climb toward a comprehensive catalog. These strategies--sequencing randomly selected cDNA clones, aligning protein sequences identified in other organisms, sequencing more genomes, and manual curation--will have to be supplemented by large-scale amplification and sequencing of specific predicted mRNAs. The steady improvements in gene prediction that have occurred over the last 10 years have increased the efficacy of this approach and decreased its cost. In this Perspective, I review the state of gene prediction roughly 10 years ago, summarize the progress that has been made since, argue that the primary ORF identification methods we have relied on so far are inadequate, and recommend a path toward completing the Catalog of Protein Coding Genes, Version 1.0. PMID:16339376

  6. C11orf95-RELA fusion present in a primary supratentorial ependymoma and recurrent sarcoma.

    PubMed

    Cachia, David; Wani, Khalida; Penas-Prado, Marta; Olar, Adriana; McCutcheon, Ian E; Benjamin, Robert S; Armstrong, Terri S; Gilbert, Mark R; Aldape, Kenneth D

    2015-04-01

    Ependymomas are rare glial tumors of the central nervous system that arise from the cells lining the ventricles and central canal within the spinal cord. The distribution of these tumors along the neuroaxis varies by age, most commonly involving the spinal cord in adults and the posterior fossa in children. It is becoming evident that ependymomas of infratentorial, supratentorial, and spinal cord location are genetically distinct which may explain the differences in clinical outcomes. A novel oncogenic fusion involving the C11orf95 and RELA genes was recently described in supratentorial ependymomas that results in constitutive aberrant activation of the nuclear factor-kB signaling pathway. Ependymosarcomas are rare neoplasms in which a malignant mesenchymal component arises within an ependymoma. We here describe a case of a sarcoma developing in a patient previously treated with chemotherapy and radiation whose original ependymoma and recurrent sarcoma were both shown to carry the type 1 C11orf95-RELA fusion transcript indicating a monoclonal origin for both tumors. PMID:25388523

  7. cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts.

    PubMed

    Damayanti, Tri Asmira; Tsukaguchi, Satoshi; Mise, Kazuyuki; Okuno, Tetsuro

    2003-09-01

    Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

  8. C9ORF72 hexanucleotide repeat expansions are a frequent cause of Huntington disease phenocopies in the Greek population.

    PubMed

    Koutsis, Georgios; Karadima, Georgia; Kartanou, Chrisoula; Kladi, Athina; Panas, Marios

    2015-01-01

    An expanded hexanucleotide repeat in C9ORF72 has been identified as the most common genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia in many populations, including the Greek. Recently, C9ORF72 expansions were reported as the most common genetic cause of Huntington disease (HD) phenocopies in a UK population. In the present study, we screened a selected cohort of 40 Greek patients with HD phenocopies for C9ORF72 hexanucleotide repeat expansions using repeat-primed polymerase chain reaction. We identified 2 patients (5%) with pathologic expansions. The first patient had chorea, behavioral-psychiatric disturbance, cognitive impairment, and a positive family history, fulfilling the strictest criteria for HD phenocopy. The second patient was sporadic and had parkinsonism, behavioral-psychiatric disturbance, and cognitive impairment, corresponding to a broader definition of HD phenocopy. These findings identify C9ORF72 expansions as a frequent cause of HD phenocopies in the Greek population, confirming recent findings in other populations and supporting proposed diagnostic testing for C9ORF72 expansions in patients with HD-like syndromes.

  9. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

    PubMed

    Donnelly, Christopher J; Zhang, Ping-Wu; Pham, Jacqueline T; Haeusler, Aaron R; Heusler, Aaron R; Mistry, Nipun A; Vidensky, Svetlana; Daley, Elizabeth L; Poth, Erin M; Hoover, Benjamin; Fines, Daniel M; Maragakis, Nicholas; Tienari, Pentti J; Petrucelli, Leonard; Traynor, Bryan J; Wang, Jiou; Rigo, Frank; Bennett, C Frank; Blackshaw, Seth; Sattler, Rita; Rothstein, Jeffrey D

    2013-10-16

    A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

  10. Comprehensive phylogenetic analysis of bacterial group II intron-encoded ORFs lacking the DNA endonuclease domain reveals new varieties.

    PubMed

    Toro, Nicolás; Martínez-Abarca, Francisco

    2013-01-01

    Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP) ORF within the ribozyme domain IV (DIV). This ORF encodes an N-terminal reverse transcriptase (RT) domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D) region followed by a DNA endonuclease (En) domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region (classes A, C, D, E, F and unclassified introns). On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain.

  11. Methylation of C9orf72 expansion reduces RNA foci formation and dipeptide-repeat proteins expression in cells.

    PubMed

    Bauer, Peter O

    2016-01-26

    A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), together referred to as c9FTD/ALS. It has been suggested that a loss of C9orf72 protein expression, the formation of toxic RNA foci and dipeptide-repeat proteins contribute to C9orf72-related diseases. Interestingly, it has been shown that trimethylation of histones and methylation of CpG islands near the repeat expansion may play a role in the pathogenesis c9FTD/ALS. Recently, methylation of expanded repeat itself has been reported. To further elucidate the mechanisms underlying these diseases, the influence of epigenetic modification in the repeat expansion on its pathogenic effect was assessed. Here, a reduced formation of toxic RNA foci and dipeptide-repeat proteins upon methylation of the GGGGCC repeat in a cellular model of c9FTD/ALS is shown. Additionally, a novel methylcytosine-capture DNA hybridization immunoassay for semi-quantitative detection of the repeat methylation levels is presented, potentially usable for methylation analysis in patients carrying C9orf72 repeat expansion carriers as a diagnostic tool. Presented results suggest that increased level of pathogenic GGGGCC expansion methylation may be sufficient to alleviate the molecular pathology of the C9orf72-related diseases.

  12. Predicted Gene Sequence C10orf112 is Transcribed, Exhibits Tissue-Specific Expression, and May Correspond to AD7

    PubMed Central

    Zubenko, George S.; Hughes, Hugh B.

    2011-01-01

    Case-control and prospective longitudinal studies have revealed an interaction of the anonymous D10S1423 234bp allele with the APOE4 allele in determining the age-specific risk of Alzheimer's Disease (AD). The D10S1423 polymorphism resides within intron 10 of open reading frame C10orf112, whose predicted product resembles a low-density lipoprotein receptor (NCBI Build 35.1). These observations suggest that the D10S1423 234bp allele may be in linkage disequilibrium with a C10orf112 gene variant whose product interacts with the apoE4 lipoprotein. Our initial exploration of this hypothesis focused on validating the C10orf112 gene model. RT-PCR amplification from human hippocampal mRNA confirmed that 34 of the predicted 39 exons of C10orf112 were expressed in this brain region. Northern blots revealed 1.2 kb and 3.2 kb mRNA species that hybridize to a cDNA probe consisting of contiguous exons 23-26. Expression of these C10orf112 mRNA species was limited to a subset of brain regions and heart tissue. PMID:19103277

  13. Soluble purified recombinant C2ORF40 protein inhibits tumor cell growth in vivo by decreasing telomerase activity in esophageal squamous cell carcinoma

    PubMed Central

    Li, Linwei; Li, Xiaoyan; Wang, Wenyu; Gao, Tianhui; Zhou, Yun; Lu, Shixin

    2016-01-01

    The chromosome 2 open reading frame 40 (C2ORF40) gene is a candidate tumor suppressor gene for a variety of tumors. Previous results by the present authors revealed that the C2ORF40 protein is a secreted protein. However, the exact biological function of secreted C2ORF40 protein in carcinogenesis has not been thoroughly investigated. In the present study, the signal peptide sequence of the C2ORF40 cDNA was initially removed to produce secreted recombinant human C2ORF40 protein (rhC2ORF40). Soluble rhC2ORF40 was successfully expressed and purified, which was evaluated for the first time, to the best of our knowledge, for tumor-suppressing function in vivo in esophageal cancer. The present results revealed that soluble purified rhC2ORF40 was concentrated with a purity of >95%. Furthermore, rhC2ORF40 inhibited esophageal cancer cell growth in vivo in a dose-dependent manner compared with a control group (P<0.05). In addition, the present study demonstrated for the first time that rhC2ORF40 decreased telomerase activity using telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (P<0.05), without affecting the expression levels of telomerase-component RNA (P>0.05), as shown with polymerase chain reaction. Overall, the present results demonstrated that soluble rhC2ORF40 inhibited tumor cell growth in vivo by decreasing telomerase activity in esophageal squamous cell carcinoma. Therefore, soluble rhC2ORF40 with a high purity and biological activity may be a potential biological therapy drug for esophageal cancer. PMID:27698864

  14. Soluble purified recombinant C2ORF40 protein inhibits tumor cell growth in vivo by decreasing telomerase activity in esophageal squamous cell carcinoma

    PubMed Central

    Li, Linwei; Li, Xiaoyan; Wang, Wenyu; Gao, Tianhui; Zhou, Yun; Lu, Shixin

    2016-01-01

    The chromosome 2 open reading frame 40 (C2ORF40) gene is a candidate tumor suppressor gene for a variety of tumors. Previous results by the present authors revealed that the C2ORF40 protein is a secreted protein. However, the exact biological function of secreted C2ORF40 protein in carcinogenesis has not been thoroughly investigated. In the present study, the signal peptide sequence of the C2ORF40 cDNA was initially removed to produce secreted recombinant human C2ORF40 protein (rhC2ORF40). Soluble rhC2ORF40 was successfully expressed and purified, which was evaluated for the first time, to the best of our knowledge, for tumor-suppressing function in vivo in esophageal cancer. The present results revealed that soluble purified rhC2ORF40 was concentrated with a purity of >95%. Furthermore, rhC2ORF40 inhibited esophageal cancer cell growth in vivo in a dose-dependent manner compared with a control group (P<0.05). In addition, the present study demonstrated for the first time that rhC2ORF40 decreased telomerase activity using telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (P<0.05), without affecting the expression levels of telomerase-component RNA (P>0.05), as shown with polymerase chain reaction. Overall, the present results demonstrated that soluble rhC2ORF40 inhibited tumor cell growth in vivo by decreasing telomerase activity in esophageal squamous cell carcinoma. Therefore, soluble rhC2ORF40 with a high purity and biological activity may be a potential biological therapy drug for esophageal cancer.

  15. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

    PubMed

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-03-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

  16. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses

    PubMed Central

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-01-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle. PMID:26884161

  17. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

    PubMed

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-03-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle. PMID:26884161

  18. Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms.

    PubMed

    Verweij, Marieke C; Wellish, Mary; Whitmer, Travis; Malouli, Daniel; Lapel, Martin; Jonjić, Stipan; Haas, Juergen G; DeFilippis, Victor R; Mahalingam, Ravi; Früh, Klaus

    2015-05-01

    Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses.

  19. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit

    PubMed Central

    He, J.; Cooper, H. M.; Reyes, A.; Di Re, M.; Kazak, L.; Wood, S. R.; Mao, C. C.; Fearnley, I. M.; Walker, J. E.; Holt, I. J.

    2012-01-01

    The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle. PMID:22447445

  20. Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin.

    PubMed

    Liu, Yanli; Liu, Shasha; Yang, Tingting; Guo, Xiaoxia; Jiang, Yali; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Yang, Jihong; Zhao, Haobin; Yang, Yi; Li, Aiying; Qi, Chao

    2015-12-01

    Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98% purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22% PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 Å resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.

  1. Amyotrophic Lateral Sclerosis with Frontotemporal Dementia in the Presence of C9orf72 Repeat Expansion-A Case Report.

    PubMed

    Bonda, Chaitanya; Kolikonda, Murali K; Brown, Martin E; Lippmann, Steven

    2016-01-01

    Amyotrophic lateral sclerosis and frontotemporal dementia are significant neurodegenerative illnesses with possible genetic predispositions. The C9orf72 gene and the GGGGCC repeat expansions of it are reported to have a causative role in the expression of these conditions. We report a case of a patient with autosomal dominant amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) in the presence of C9orf72 repeat expansion. We believe our case further supports the theory that the presence of C9orf72 repeat expansion in patients with a family history of amyotrophic lateral sclerosis and/or frontotemporal dementia significantly increases their risk of developing either or both diseases. The development of antisense oligonucleotides that might target GGGGCC RNA sequences theoretically may have a therapeutic role in mitigating the clinical expression of these illnesses.

  2. Tumorigenic poxviruses: genomic organization and DNA sequence of the telomeric region of the Shope fibroma virus genome.

    PubMed

    Upton, C; DeLange, A M; McFadden, G

    1987-09-01

    Shope fibroma virus (SFV), a tumorigenic poxvirus, has a 160-kb linear double-stranded DNA genome and possesses terminal inverted repeats (TIRs) of 12.4 kb. The DNA sequence of the terminal 5.5 kb of the viral genome is presented and together with previously published sequences completes the entire sequence of the SFV TIR. The terminal 400-bp region contains no major open reading frames (ORFs) but does possess five related imperfect palindromes. The remaining 5.1 kb of the sequence contains seven tightly clustered and tandemly oriented ORFs, four larger than 100 amino acids in length (T1, T2, T4, and T5) and three smaller ORFs (T3A, T3B, and T3C). All are transcribed toward the viral hairpin and almost all possess the consensus sequence TTTTTNT near their 3' ends which has been implicated for the transcription termination of vaccinia virus early genes. Searches of the published DNA database revealed no sequences with significant homology with this region of the SFV genome but when the protein database was searched with the translation products of ORFs T1-T5 it was found that the N-terminus of the putative T4 polypeptide is closely related to the signal sequence of the hemagglutinin precursor from influenza A virus, suggesting that the T4 polypeptide may be secreted from SFV-infected cells. Examination of other SFV ORFs shows that T1 and T2 also possess signal-like hydrophobic amino acid stretches close to their N-termini. The protein database search also revealed that the putative T2 protein has significant homology to the insulin family of polypeptides. In terms of sequence repetitions, seven tandemly repeated copies of the hexanucleotide ATTGTT and three flanking regions of dyad symmetry were detected, all in ORF T3C. A search for palindromic sequences also revealed two clusters, one in ORF T3A/B and a second in ORF T2. ORF T2 harbors five short sequence domains, each of which consists of a 6-bp short palindrome and a 10- to 18-bp larger palindrome. The

  3. The Footprint of Genome Architecture in the Largest Genome Expansion in RNA Viruses

    PubMed Central

    Lauber, Chris; Goeman, Jelle J.; Parquet, Maria del Carmen; Thi Nga, Phan; Snijder, Eric J.; Morita, Kouichi; Gorbalenya, Alexander E.

    2013-01-01

    The small size of RNA virus genomes (2-to-32 kb) has been attributed to high mutation rates during replication, which is thought to lack proof-reading. This paradigm is being revisited owing to the discovery of a 3′-to-5′ exoribonuclease (ExoN) in nidoviruses, a monophyletic group of positive-stranded RNA viruses with a conserved genome architecture. ExoN, a homolog of canonical DNA proof-reading enzymes, is exclusively encoded by nidoviruses with genomes larger than 20 kb. All other known non-segmented RNA viruses have smaller genomes. Here we use evolutionary analyses to show that the two- to three-fold expansion of the nidovirus genome was accompanied by a large number of replacements in conserved proteins at a scale comparable to that in the Tree of Life. To unravel common evolutionary patterns in such genetically diverse viruses, we established the relation between genomic regions in nidoviruses in a sequence alignment-free manner. We exploited the conservation of the genome architecture to partition each genome into five non-overlapping regions: 5′ untranslated region (UTR), open reading frame (ORF) 1a, ORF1b, 3′ORFs (encompassing the 3′-proximal ORFs), and 3′ UTR. Each region was analyzed for its contribution to genome size change under different models. The non-linear model statistically outperformed the linear one and captured >92% of data variation. Accordingly, nidovirus genomes were concluded to have reached different points on an expansion trajectory dominated by consecutive increases of ORF1b, ORF1a, and 3′ORFs. Our findings indicate a unidirectional hierarchical relation between these genome regions, which are distinguished by their expression mechanism. In contrast, these regions cooperate bi-directionally on a functional level in the virus life cycle, in which they predominantly control genome replication, genome expression, and virus dissemination, respectively. Collectively, our findings suggest that genome architecture and the

  4. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration

    PubMed Central

    Lagier-Tourenne, Clotilde; Baughn, Michael; Rigo, Frank; Sun, Shuying; Liu, Patrick; Li, Hai-Ri; Jiang, Jie; Watt, Andrew T.; Chun, Seung; Katz, Melanie; Qiu, Jinsong; Sun, Ying; Ling, Shuo-Chien; Zhu, Qiang; Polymenidou, Magdalini; Drenner, Kevin; Artates, Jonathan W.; McAlonis-Downes, Melissa; Markmiller, Sebastian; Hutt, Kasey R.; Pizzo, Donald P.; Cady, Janet; Harms, Matthew B.; Baloh, Robert H.; Vandenberg, Scott R.; Yeo, Gene W.; Fu, Xiang-Dong; Bennett, C. Frank; Cleveland, Don W.; Ravits, John

    2013-01-01

    Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions. PMID:24170860

  5. C9orf72 mutation is rare in Alzheimer's disease, Parkinson's disease, and essential tremor in China

    PubMed Central

    Jiao, Bin; Guo, Ji-feng; Wang, Ya-qin; Yan, Xin-xiang; Zhou, Lin; Liu, Xiao-yan; Zhang, Fu-feng; Zhou, Ya-fang; Xia, Kun; Tang, Bei-sha; Shen, Lu

    2013-01-01

    GGGGCC repeat expansions in the C9orf72 gene have been identified as a major contributing factor in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the overlapping of clinical phenotypes and pathological characteristics between these two diseases and Alzheimer's disease (AD), Parkinson's disease (PD), and essential tremor (ET), we speculated regarding whether C9orf72 repeat expansions also play a major role in these three diseases. Using the repeat-primed polymerase chain reaction method, we screened for C9orf72 in three groups of patients with PD (n = 911), AD (n = 279), and ET (n = 152) in the Chinese Han population. There were no pathogenic repeats (>30 repeats) detected in either the patients or controls (n = 314), which indicated that the pathogenic expansions of C9orf72 might be rare in these three diseases. However, the analysis of the association between the number of repeats (p = 0.001), short/intermediate genotype (short: <7 repeats; intermediate: ≥7 repeats) (odds ratio 1.37 [1.05, 1.79]), intermediate/intermediate genotype (Odds ratio 2.03 [1.17, 3.54]), and PD risks indicated that intermediate repeat alleles could act as contributors to PD. To the best of our knowledge, this study is the first to reveal the correlation between C9orf72 and Chinese PD, AD, or ET patients. Additionally, the results of this study suggest the novel idea that the intermediate repeat allele in C9orf72 is most likely a risk factor for PD. PMID:24068985

  6. TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions.

    PubMed

    Gallagher, Michael D; Suh, Eunran; Grossman, Murray; Elman, Lauren; McCluskey, Leo; Van Swieten, John C; Al-Sarraj, Safa; Neumann, Manuela; Gelpi, Ellen; Ghetti, Bernardino; Rohrer, Jonathan D; Halliday, Glenda; Van Broeckhoven, Christine; Seilhean, Danielle; Shaw, Pamela J; Frosch, Matthew P; Alafuzoff, Irina; Antonell, Anna; Bogdanovic, Nenad; Brooks, William; Cairns, Nigel J; Cooper-Knock, Johnathan; Cotman, Carl; Cras, Patrick; Cruts, Marc; De Deyn, Peter P; DeCarli, Charles; Dobson-Stone, Carol; Engelborghs, Sebastiaan; Fox, Nick; Galasko, Douglas; Gearing, Marla; Gijselinck, Ilse; Grafman, Jordan; Hartikainen, Päivi; Hatanpaa, Kimmo J; Highley, J Robin; Hodges, John; Hulette, Christine; Ince, Paul G; Jin, Lee-Way; Kirby, Janine; Kofler, Julia; Kril, Jillian; Kwok, John B J; Levey, Allan; Lieberman, Andrew; Llado, Albert; Martin, Jean-Jacques; Masliah, Eliezer; McDermott, Christopher J; McKee, Ann; McLean, Catriona; Mead, Simon; Miller, Carol A; Miller, Josh; Munoz, David G; Murrell, Jill; Paulson, Henry; Piguet, Olivier; Rossor, Martin; Sanchez-Valle, Raquel; Sano, Mary; Schneider, Julie; Silbert, Lisa C; Spina, Salvatore; van der Zee, Julie; Van Langenhove, Tim; Warren, Jason; Wharton, Stephen B; White, Charles L; Woltjer, Randall L; Trojanowski, John Q; Lee, Virginia M Y; Van Deerlin, Vivianna; Chen-Plotkin, Alice S

    2014-03-01

    Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently been linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis, and may be the most common genetic cause of both neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the most common neuropathological subtype of FTLD, characterized by inclusions of TAR DNA-binding protein of 43 kDa (FTLD-TDP). Previous reports have shown that TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN) mutations, with the major (risk) allele of rs1990622 associating with earlier age at onset of disease. Here, we report that rs1990622 genotype affects age at death in a single-site discovery cohort of FTLD patients with C9orf72 expansions (n = 14), with the major allele correlated with later age at death (p = 0.024). We replicate this modifier effect in a 30-site international neuropathological cohort of FTLD-TDP patients with C9orf72 expansions (n = 75), again finding that the major allele associates with later age at death (p = 0.016), as well as later age at onset (p = 0.019). In contrast, TMEM106B genotype does not affect age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72 expansions. Intriguingly, the genotype that confers increased risk for developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later age at onset and death in C9orf72 expansion carriers, providing an example of sign epistasis in human neurodegenerative disease.

  7. A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

    PubMed Central

    Zhang, Rui; Liu, Shengxue; Chiba, Sotaro; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2014-01-01

    Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5′-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1. PMID:25101066

  8. Cardiomyopathy Syndrome of Atlantic Salmon (Salmo salar L.) Is Caused by a Double-Stranded RNA Virus of the Totiviridae Family▿

    PubMed Central

    Haugland, Øyvind; Mikalsen, Aase B.; Nilsen, Pål; Lindmo, Karine; Thu, Beate J.; Eliassen, Trygve M.; Roos, Norbert; Rode, Marit; Evensen, Øystein

    2011-01-01

    Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease. PMID:21411528

  9. c21orf59/kurly Controls Both Cilia Motility and Polarization.

    PubMed

    Jaffe, Kimberly M; Grimes, Daniel T; Schottenfeld-Roames, Jodi; Werner, Michael E; Ku, Tse-Shuen J; Kim, Sun K; Pelliccia, Jose L; Morante, Nicholas F C; Mitchell, Brian J; Burdine, Rebecca D

    2016-03-01

    Cilia are microtubule-based projections that function in the movement of extracellular fluid. This requires cilia to be: (1) motile and driven by dynein complexes and (2) correctly polarized on the surface of cells, which requires planar cell polarity (PCP). Few factors that regulate both processes have been discovered. We reveal that C21orf59/Kurly (Kur), a cytoplasmic protein with some enrichment at the base of cilia, is needed for motility; zebrafish mutants exhibit characteristic developmental abnormalities and dynein arm defects. kur was also required for proper cilia polarization in the zebrafish kidney and the larval skin of Xenopus laevis. CRISPR/Cas9 coupled with homologous recombination to disrupt the endogenous kur locus in Xenopus resulted in the asymmetric localization of the PCP protein Prickle2 being lost in mutant multiciliated cells. Kur also makes interactions with other PCP components, including Disheveled. This supports a model wherein Kur plays a dual role in cilia motility and polarization.

  10. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

    PubMed

    Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie; Almeida, Sandra; Cook, Casey N; Gendron, Tania F; Prudencio, Mercedes; Van Blitterswijk, Marka; Belzil, Veronique; Couthouis, Julien; Paul, Joseph West; Goodman, Lindsey D; Daughrity, Lillian; Chew, Jeannie; Garrett, Aliesha; Pregent, Luc; Jansen-West, Karen; Tabassian, Lilia J; Rademakers, Rosa; Boylan, Kevin; Graff-Radford, Neill R; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Boeve, Bradley F; Deng, Ning; Feng, Yanan; Cheng, Tzu-Hao; Dickson, Dennis W; Cohen, Stanley N; Bonini, Nancy M; Link, Christopher D; Gao, Fen-Biao; Petrucelli, Leonard; Gitler, Aaron D

    2016-08-12

    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately. PMID:27516603

  11. Deep Sequencing Analysis of Apple Infecting Viruses in Korea

    PubMed Central

    Cho, In-Sook; Igori, Davaajargal; Lim, Seungmo; Choi, Gug-Seoun; Hammond, John; Lim, Hyoun-Sub; Moon, Jae Sun

    2016-01-01

    Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time. PMID:27721694

  12. The Kaposi's sarcoma-associated herpesvirus ORF34 protein binds to HIF-1α and causes its degradation via the proteasome pathway.

    PubMed

    Haque, Muzammel; Kousoulas, Konstantin G

    2013-02-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761-6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway. PMID:23221556

  13. GGGGCC repeat expansion in C9ORF72 compromises nucleocytoplasmic transport

    PubMed Central

    Freibaum, Brian D.; Lu, Yubing; Lopez-Gonzalez, Rodrigo; Kim, Nam Chul; Almeida, Sandra; Lee, Kyung-Ha; Badders, Nisha; Valentine, Marc; Miller, Bruce L.; Wong, Philip C.; Petrucelli, Leonard; Kim, Hong Joo; Gao, Fen-Biao; Taylor, J. Paul

    2015-01-01

    GGGGCC (G4C2) repeat expansion in a noncoding region of C9ORF72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1,2. The basis for pathogenesis is unknown. To capture the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2 repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein (GFP) to detect repeat-associated non-AUG (RAN) translation. These transgenic animals show dosage-dependent, repeat length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins as observed in patients. This model was used in a large-scale, unbiased genetic screen ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC) as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged iPSC-derived neurons from C9ORF72 patients. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration. PMID:26308899

  14. Whole-Genome Sequences of Novel Porcine Circovirus Type 2 Viruses Detected in Swine from Mexico and the United States.

    PubMed

    Harmon, Karen M; Gauger, Phillip C; Zhang, Jianqiang; Piñeyro, Pablo E; Dunn, Derek D; Chriswell, Amy J

    2015-01-01

    A unique porcine circovirus type 2 capsid protein (ORF2) sequence was detected in swine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. The complete genome sequences of four viruses, one from Mexico and three from the United States, were determined to further characterize this novel PCV2 genotype. PMID:26679573

  15. Whole-Genome Sequences of Novel Porcine Circovirus Type 2 Viruses Detected in Swine from Mexico and the United States

    PubMed Central

    Gauger, Phillip C.; Zhang, Jianqiang; Piñeyro, Pablo E.; Dunn, Derek D.; Chriswell, Amy J.

    2015-01-01

    A unique porcine circovirus type 2 capsid protein (ORF2) sequence was detected in swine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. The complete genome sequences of four viruses, one from Mexico and three from the United States, were determined to further characterize this novel PCV2 genotype. PMID:26679573

  16. Complete Genome Sequence of an NADC30-Like Porcine Reproductive and Respiratory Syndrome Virus Characterized by Recombination with Other Strains

    PubMed Central

    Li, Yingying; Ji, Guobiao; Wang, Juan; Tan, Feifei; Zhuang, Jinshan

    2016-01-01

    We report here the complete genome sequence of an NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV), HNyc15, which was characterized by recombination with VR-2332 and CH-1a PRRSV strains in open reading frames (ORFs) 2 to 4. PMID:27151798

  17. Beet western yellows virus infects the carnivorous plant Nepenthes mirabilis.

    PubMed

    Miguel, Sissi; Biteau, Flore; Mignard, Benoit; Marais, Armelle; Candresse, Thierry; Theil, Sébastien; Bourgaud, Frédéric; Hehn, Alain

    2016-08-01

    Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5.

  18. Beet western yellows virus infects the carnivorous plant Nepenthes mirabilis.

    PubMed

    Miguel, Sissi; Biteau, Flore; Mignard, Benoit; Marais, Armelle; Candresse, Thierry; Theil, Sébastien; Bourgaud, Frédéric; Hehn, Alain

    2016-08-01

    Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5. PMID:27180098

  19. Recombination and natural selection in hepatitis E virus genotypes.

    PubMed

    Chen, Xiaoming; Zhang, Qian; He, Chao; Zhang, Lei; Li, Jinghua; Zhang, Weilu; Cao, Wei; Lv, Yong-Gang; Liu, Zhengcai; Zhang, Jing-Xia; Shao, Zhong-Jun

    2012-09-01

    To gain new insights into the evolutionary processes that created the genetic diversity of the hepatitis E virus (HEV), the Recombination Detection Program (RDP) and SimPlot program were employed to detect recombination events in the genome, then the fixed-effects likelihood (FEL) method was used to detect natural selection effects on viral proteins. Recombination analysis provided strong evidence for both intergenotype and intragenotype recombination events in the sequences analyzed. Recombination events were found to be distributed non-randomly, with the highest frequency in the X domain and the helicase. Strain DQ450072 was identified as intergenotype-recombinant. Natural selection analysis revealed that codons under both negative selection and positive selection were distributed non-randomly. ORF1 and ORF2 have experienced strong purifying selection across genotypes. Furthermore, potentially important sites were also found under positive selection in the N-terminal end of ORF2 and the C-terminal end of ORF3. No significant difference was found among the selective pressures on different genotypes.

  20. Plant genomes enclose footprints of past infections by giant virus relatives.

    PubMed

    Maumus, Florian; Epert, Aline; Nogué, Fabien; Blanc, Guillaume

    2014-01-01

    Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb-2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants.

  1. Novel DNA virus isolated from samples showing endothelial cell necrosis in the Japanese eel, Anguilla japonica.

    PubMed

    Mizutani, Tetsuya; Sayama, Yusuke; Nakanishi, Akira; Ochiai, Hideharu; Sakai, Kouji; Wakabayashi, Kouji; Tanaka, Nozomi; Miura, Emi; Oba, Mami; Kurane, Ichiro; Saijo, Masayuki; Morikawa, Shigeru; Ono, Shin-ichi

    2011-03-30

    Economic loss due to viral endothelial cell necrosis of eel (VECNE) of Anguilla japonica is a serious problem for the cultured Japanese eel market. However, the viral genome responsible for VECNE is unknown. We recently developed a rapid determination system for viral nucleic acid sequences (RDV) to determine viral genome sequences. In this study, viral DNA fragments were obtained using RDV, and approximately 15-kbp circular full genome sequences were determined using a next-generation sequencing system, overlapping PCR, and Southern blot analysis. One open reading frame (ORF) was homologous to the large T-antigen of polyomavirus; other ORFs have no homology with any nucleic or amino acid sequences of polyomavirus. Therefore, as this DNA virus might comprise a novel virus family, we provisionally named it Japanese eel endothelial cells-infecting virus (JEECV). JEECV was detected in both naturally and experimentally infected eels, suggesting that JEECV potentially causes VECNE. PMID:21277610

  2. Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia.

    PubMed

    Austin-Tse, Christina; Halbritter, Jan; Zariwala, Maimoona A; Gilberti, Renée M; Gee, Heon Yung; Hellman, Nathan; Pathak, Narendra; Liu, Yan; Panizzi, Jennifer R; Patel-King, Ramila S; Tritschler, Douglas; Bower, Raqual; O'Toole, Eileen; Porath, Jonathan D; Hurd, Toby W; Chaki, Moumita; Diaz, Katrina A; Kohl, Stefan; Lovric, Svjetlana; Hwang, Daw-Yang; Braun, Daniela A; Schueler, Markus; Airik, Rannar; Otto, Edgar A; Leigh, Margaret W; Noone, Peadar G; Carson, Johnny L; Davis, Stephanie D; Pittman, Jessica E; Ferkol, Thomas W; Atkinson, Jeffry J; Olivier, Kenneth N; Sagel, Scott D; Dell, Sharon D; Rosenfeld, Margaret; Milla, Carlos E; Loges, Niki T; Omran, Heymut; Porter, Mary E; King, Stephen M; Knowles, Michael R; Drummond, Iain A; Hildebrandt, Friedhelm

    2013-10-01

    Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

  3. Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

    PubMed Central

    Austin-Tse, Christina; Halbritter, Jan; Zariwala, Maimoona A.; Gilberti, Renée M.; Gee, Heon Yung; Hellman, Nathan; Pathak, Narendra; Liu, Yan; Panizzi, Jennifer R.; Patel-King, Ramila S.; Tritschler, Douglas; Bower, Raqual; O’Toole, Eileen; Porath, Jonathan D.; Hurd, Toby W.; Chaki, Moumita; Diaz, Katrina A.; Kohl, Stefan; Lovric, Svjetlana; Hwang, Daw-Yang; Braun, Daniela A.; Schueler, Markus; Airik, Rannar; Otto, Edgar A.; Leigh, Margaret W.; Noone, Peadar G.; Carson, Johnny L.; Davis, Stephanie D.; Pittman, Jessica E.; Ferkol, Thomas W.; Atkinson, Jeffry J.; Olivier, Kenneth N.; Sagel, Scott D.; Dell, Sharon D.; Rosenfeld, Margaret; Milla, Carlos E.; Loges, Niki T.; Omran, Heymut; Porter, Mary E.; King, Stephen M.; Knowles, Michael R.; Drummond, Iain A.; Hildebrandt, Friedhelm

    2013-01-01

    Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65. PMID:24094744

  4. LINE-1 ORF-1p functions as a novel HGF/ETS-1 signaling pathway co-activator and promotes the growth of MDA-MB-231 cell.

    PubMed

    Yang, Qian; Feng, Fan; Zhang, Fan; Wang, Chunping; Lu, Yinying; Gao, Xudong; Zhu, Yunfeng; Yang, Yongping

    2013-12-01

    Long interspersed nucleotide element (LINE)-1 ORF-1p is encoded by the human pro-oncogene LINE-1. It is involved in the development and progression of several human carcinomas, such as hepatocellular carcinoma and lung and breast cancers. The hepatocyte growth factor (HGF)/ETS-1 signaling pathway is involved in regulation of cancer cell proliferation, metastasis and invasion. The biological function of the interaction between LINE-1 ORF-1p and the HGF/ETS-1 signaling pathway in regulation of human breast cancer proliferation remains largely unknown. Here, we showed that LINE-1 ORF-1p enhanced ETS-1 transcriptional activity and increased expression of downstream genes of ETS-1. Interaction between ETS-1 and LINE-1 ORF-1p was identified by immunoprecipitation assays. LINE-1 ORF-1p modulated ETS-1 activity through cytoplasm/nucleus translocation and recruitment to the ETS-1 binding element in the MMP1 gene promoter. We also showed that LINE-1 ORF-1p promoted proliferation and anchorage-independent growth of MDA-MB-231 breast cancer cells. By investigating a novel role of the LINE-1 ORF-1p in the HGF/ETS-1 signaling pathway and MDA-MB-231 cells, we demonstrated that LINE-1 ORF-1p may be a novel ETS-1 coactivator and molecular target for therapy of human triple negative breast cancer.

  5. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

    PubMed Central

    de Munnik, Sabrina M.; Kooistra, Albert J.; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi’s sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74. PMID:25894435

  6. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

    PubMed

    de Munnik, Sabrina M; Kooistra, Albert J; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J; Leurs, Rob; Vischer, Henry F

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

  7. Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12.

    PubMed

    Poteete, Anthony R

    2004-05-01

    The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants. PMID:15090511

  8. ALS/FTD phenotype in two Sardinian families carrying both C9ORF72 and TARDBP mutations

    PubMed Central

    Chiò, Adriano; Restagno, Gabriella; Brunetti, Maura; Ossola, Irene; Calvo, Andrea; Canosa, Antonio; Moglia, Cristina; Floris, Gianluca; Tacconi, Paolo; Marrosu, Francesco; Marrosu, Maria Giovanna; Murru, Maria Rita; Majounie, Elisa; Renton, Alan E; Abramzon, Yvegeniya; Pugliatti, Maura; Sotgiu, Maria Alessandra; Traynor, Bryan J; Borghero, Giuseppe

    2015-01-01

    Background In the isolated population of Sardinia, a Mediterranean island, ~25% of ALS cases carry either a p.A382T mutation of the TARDBP gene or a GGGGCC hexanucleotide repeat expansion in the first intron of the C9ORF72 gene. Objective To describe the co-presence of two genetic mutations in two Sardinian ALS patients. Methods We identified two index ALS cases carrying both the p.A382T missense mutation of TARDBP gene and the hexanucleotide repeat expansion of C9ORF72 gene. Results The index case of Family A had bulbar ALS and frontemporal dementia (FTD) at 43. His father, who carried the hexanucleotide repeat expansion of C9ORF72 gene, had spinal ALS and FTD at 64 and his mother, who carried the TARDBP gene p.A382T missense mutation, had spinal ALS and FTD at 69. The index case of Family B developed spinal ALS without FTD at 35 and had a rapid course to respiratory failure. His parents are healthy at 62 and 63. The two patients share the known founder risk haplotypes across both the C9ORF72 9p21 locus and the TARDBP 1p36.22 locus. Conclusions Our data show that in rare neurodegenerative causing genes can co-exist within the same individuals and are associated with a more severe disease course. PMID:22550220

  9. X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15.

    PubMed

    Demirci, F Yesim K; Rigatti, Brian W; Wen, Gaiping; Radak, Amy L; Mah, Tammy S; Baic, Corrine L; Traboulsi, Elias I; Alitalo, Tiina; Ramser, Juliane; Gorin, Michael B

    2002-04-01

    X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.

  10. The complete nucleotide sequence and genome organization of pea streak virus (genus Carlavirus).

    PubMed

    Su, Li; Li, Zhengnan; Bernardy, Mike; Wiersma, Paul A; Cheng, Zhihui; Xiang, Yu

    2015-10-01

    Pea streak virus (PeSV) is a member of the genus Carlavirus in the family Betaflexiviridae. Here, the first complete genome sequence of PeSV was determined by deep sequencing of a cDNA library constructed from dsRNA extracted from a PeSV-infected sample and Rapid Amplification of cDNA Ends (RACE) PCR. The PeSV genome consists of 8041 nucleotides excluding the poly(A) tail and contains six open reading frames (ORFs). The putative peptide encoded by the PeSV ORF6 has an estimated molecular mass of 6.6 kDa and shows no similarity to any known proteins. This differs from typical carlaviruses, whose ORF6 encodes a 12- to 18-kDa cysteine-rich nucleic-acid-binding protein.

  11. Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

    SciTech Connect

    Liu Wangjing; Chang Yunshiang; Wang Chunghsiung; Kou, Guang-Hsiung; Lo Chufang . E-mail: gracelow@ntu.edu.tw

    2005-04-10

    Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

  12. Comparison of the sequence of the secretory glycoprotein A (gA) gene in Md5 and BC-1 strains of Marek's disease virus type 1.

    PubMed

    Ihara, T; Kato, A; Ueda, S; Ishihama, A; Hirai, K

    1989-11-01

    DNA fragments containing the secretory glycoprotein A (gA) gene of Marek's disease virus type 1 (MDV1) were cloned from the DNA libraries of very virulent Md5 and virulent BC-1 strains and sequenced. Two open reading frames (ORF1 and ORF2) were identified for both strains. The ORF1 has the potential to code for a protein of 501 amino acids with a molecular weight of 56 kD that contains strong hydrophobic regions in both the amino and carboxyl termini, and nine potential N-linked glycosylation sites, while the ORF2 is capable of coding for a 24-kD protein. These results indicate that the ORF1 codes for the unprocessed form of gA. Between the Md5 and BC-1 strains, only two sequence mismatches exist in the DNA fragment. More differences appear to exist in the gA sequence of the MDV1 GA strain (12), which lacks a strong hydrophobic anchor sequence. Similarities between the predicted amino acid sequences of the MDV1 gA and the proteins of the other herpesviruses such as herpes simplex type I gC, pseudorabies virus gIII, and varicella zoster virus gpV were noted.

  13. Rhabdovirus-like endogenous viral elements in the genome of Spodoptera frugiperda insect cells are actively transcribed: Implications for adventitious virus detection.

    PubMed

    Geisler, Christoph; Jarvis, Donald L

    2016-07-01

    Spodoptera frugiperda (Sf) cell lines are used to produce several biologicals for human and veterinary use. Recently, it was discovered that all tested Sf cell lines are persistently infected with Sf-rhabdovirus, a novel rhabdovirus. As part of an effort to search for other adventitious viruses, we searched the Sf cell genome and transcriptome for sequences related to Sf-rhabdovirus. To our surprise, we found intact Sf-rhabdovirus N- and P-like ORFs, and partial Sf-rhabdovirus G- and L-like ORFs. The transcribed and genomic sequences matched, indicating the transcripts were derived from the genomic sequences. These appear to be endogenous viral elements (EVEs), which result from the integration of partial viral genetic material into the host cell genome. It is theoretically impossible for the Sf-rhabdovirus-like EVEs to produce infectious virus particles as 1) they are disseminated across 4 genomic loci, 2) the G and L ORFs are incomplete, and 3) the M ORF is missing. Our finding of transcribed virus-like sequences in Sf cells underscores that MPS-based searches for adventitious viruses in cell substrates used to manufacture biologics should take into account both genomic and transcribed sequences to facilitate the identification of transcribed EVE's, and to avoid false positive detection of replication-competent adventitious viruses.

  14. Association of the plasma riboflavin levels and riboflavin transporter (C20orf54) gene statuses in Kazak esophageal squamous cell carcinoma patients.

    PubMed

    Ainiwaer, Julaiti; Tuerhong, Abuduaini; Hasim, Ayshamgul; Chengsong, Du; Liwei, Zhang; Sheyhidin, Ilyar

    2013-05-01

    To evaluate the association of the plasma riboflavin level in Kazak esophageal cancer patients and their riboflavin transporter (C20orf54) gene statuses. Plasma riboflavin levels were detected by high performance liquid chromatography in Kazak patients with esophageal squamous cell carcinoma (ESCC) and healthy controls. C20orf54 mRNA and protein expression were analyzed by real-time fluorogenic quantitative polymerase chain reaction and immunohistochemistry in samples from 61 ESCC patients consisting of both tumor and normal tissue, respectively. C20orf54 mRNA expression was decreased in ESCC (0.279 ± 0.102) than in normal counterpart tissue (0.479 ± 0.287; P = 0.049) significantly. Tumors exhibited low C20orf54 protein expression (42.6, 26.2, 18.0 and 13.1% for no C20orf54 staining, weak staining, medium staining and strong staining, respectively), which was significantly lower than that in the normal mucous membrane (13.1, 26.2, 41.0 and 19.7% for no C20orf54 staining, weak staining, medium staining and strong staining, respectively). Defective expression of C20orf54 in tumor cells was significantly associated with poor differentiation. However, other parameters such as depth of invasion and lymph node metastasis had no significant relationship with C20orf54 expression. The average blood concentration of riboflavin was 2.6468 ± 1.3474 ng/ml in ESCC patients lower than control group (4.2960 ± 3.2293 ng/ml, P = 0.015). A positive correlation of plasma riboflavin levels with defective expression of C20orf54 protein was found in ESCC patients (F = 8.626; P = 0.038). Defective expression of C20orf54 is associated with the development of Kazak esophageal squamous cell carcinoma and this may represent a mechanism underlying the decreased plasma riboflavin levels in ESCC.

  15. E4orf4 induces PP2A- and Src-dependent cell death in Drosophila melanogaster and at the same time inhibits classic apoptosis pathways

    PubMed Central

    Pechkovsky, Antonina; Lahav, Maoz; Bitman, Eliya; Salzberg, Adi; Kleinberger, Tamar

    2013-01-01

    The adenovirus E4orf4 protein regulates the progression of viral infection, and when expressed alone in mammalian tissue culture cells it induces protein phosphatase 2A (PP2A)-B55– and Src-dependent cell death, which is more efficient in oncogene-transformed cells than in normal cells. This form of cell death is caspase-independent, although it interacts with classic caspase-dependent apoptosis. PP2A-B55–dependent E4orf4-induced toxicity is highly conserved in evolution from yeast to mammalian cells. In this work we investigated E4orf4-induced cell death in a whole multicellular organism, Drosophila melanogaster. We show that E4orf4 induced low levels of cell killing, caused by both caspase-dependent and -independent mechanisms. Drosophila PP2A-B55 (twins/abnormal anaphase resolution) and Src64B contributed additively to this form of cell death. Our results provide insight into E4orf4-induced cell death, demonstrating that in parallel to activating caspase-dependent apoptosis, E4orf4 also inhibited this form of cell death induced by the proapoptotic genes reaper, head involution defective, and grim. The combination of both induction and inhibition of caspase-dependent cell death resulted in low levels of tissue damage that may explain the inefficient cell killing induced by E4orf4 in normal cells in tissue culture. Furthermore, E4orf4 inhibited JNK-dependent cell killing as well. However, JNK inhibition did not impede E4orf4-induced toxicity and even enhanced it, indicating that E4orf4