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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. Novel immune-modulator identified by a rapid, functional screen of the parapoxvirus ovis (Orf virus) genome

    PubMed Central

    2012-01-01

    Background The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach. Results The genome of Parapoxvirus ovis (Orf virus) was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer. Conclusion A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration of B2 may provide the

  4. Orf virus infection in sheep or goats.

    PubMed

    Spyrou, V; Valiakos, G

    2015-12-14

    Orf virus, a member of the genus Parapoxvirus, is the causative agent of contagious ecthyma ('Orf'). It is a pathogen with worldwide distribution, causing significant financial losses in livestock production. The disease mainly affects sheep and goats, but various other ruminants and mammals have been reported to be infected as well. It is also a zoonotic disease, affecting mainly people who come in direct or indirect contact with infected animals (e.g. farmers, veterinarians). The disease is usually benign and self-limiting, although in many cases, especially in young animals, it can be persistent and even fatal. Production losses caused by Orf virus are believed to be underestimated, as it is not a notifiable disease. This review of literature presents all latest information regarding the virus; considerations regarding treatment and prevention will be also discussed. PMID:26315771

  5. Diagnosis of bovine-associated parapoxvirus infections in humans: molecular and epidemiological evidence.

    PubMed

    MacNeil, A; Lederman, E; Reynolds, M G; Ragade, N J; Talken, R; Friedman, D; Hall, W; Shwe, T; Li, Y; Zhao, H; Smith, S; Davidson, W; Hughes, C; Damon, I K

    2010-12-01

    Orf virus, pseudocowpox virus and bovine papular stomatitis virus, are parapoxviruses, associated with domestic ruminants, which are capable of causing cutaneous infections in humans. Owing to virtually identical appearances in humans, clinical differentiation of these viruses is difficult. We discuss three recent occurrences of parapoxvirus infection, involving contact with domestic bovine and use a combination of molecular and epidemiological data in the diagnosis. These cases underscore the utility of modern diagnostic tools, along with species-specific contact information in acquiring a definitive diagnosis, in the case of suspected parapoxvirus infection. PMID:20163577

  6. 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

  7. 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

  8. Specific qPCR assays for the detection of orf virus, pseudocowpox virus and bovine papular stomatitis virus.

    PubMed

    Zhao, Hui; Wilkins, Kimberly; Damon, Inger K; Li, Yu

    2013-12-01

    The genus Parapoxvirus (PAPV) is comprised traditionally of orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV), which cause infections of ruminants and their handlers in the U.S. and worldwide. Unlike orthopoxvirus infections, which can cause systemic or localized infections, PAPV infections present normally as benign, self-limited and localized skin lesions; infections do not confer lifelong immunity. In recent years, related potentially to enhanced awareness and the availability of diagnostic methods, there has been an observed increase in reported cases of PAPV in animals and humans. This study describes TaqMan based real-time PCR assays for both generic and specific detection of PAPV species for surveillance and outbreak investigations. These assays target highly conserved PAPV RNA polymerase gene sequences and are capable of detecting three known species of PAPVs (ORFV, PCPV, and BPSV). The assays were evaluated using a panel of PAPV DNA derived from human infections or animal specimen remainders. The sensitivities of all four assays were determined using droplet digital PCR; fewer than 10 copies of clinical PAPV DNA can be detected consistently. These assays provide a reliable and sensitive method for rapid confirmation and characterization PAPV infections with varying clinical presentations. PMID:24035807

  9. 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

  10. Surveillance of parapoxvirus among ruminants in Virginia and Connecticut.

    PubMed

    Roess, A A; McCollum, A M; Gruszynski, K; Zhao, H; Davidson, W; Lafon, N; Engelmeyer, T; Moyer, B; Godfrey, C; Kilpatrick, H; Labonte, A; Murphy, J; Carroll, D S; Li, Y; Damon, I K

    2013-12-01

    In 2008, two deer hunters in Virginia and Connecticut were infected with a unique strain of pseudocowpox virus, a parapoxvirus. To estimate the prevalence of this virus, and in an attempt to define the reservoir, Parapoxvirus surveillance was undertaken between November 2009 and January 2010. 125 samples from four ruminant species (cows, goat, sheep and white-tailed deer) were collected in Virginia, and nine samples from white-tailed deer were collected in Connecticut. We found no evidence that the parapoxvirus species that infected the deer hunters is circulating among domesticated ruminants or white-tailed deer. However, parapoxvirus DNA of a different parapoxvirus species, bovine papular stomatitis virus (BPSV), was detected in 31 samples obtained from asymptomatic cattle in Virginia. Parapoxvirus DNA-positive cattle originated from the same counties indicating probable transmission among animals. Molecular analysis identified BPSV as the parapoxvirus affecting animals. Asymptomatic parapoxvirus infections in livestock, particularly young animals, may be common, and further investigation will inform our knowledge of virus transmission. PMID:23398718

  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. Orf virus IL-10 reduces monocyte, dendritic cell and mast cell recruitment to inflamed skin.

    PubMed

    Bennett, Jared R; Lateef, Zabeen; Fleming, Stephen B; Mercer, Andrew A; Wise, Lyn M

    2016-02-01

    Orf virus (ORFV) is a zoonotic parapoxvirus that causes pustular dermatitis of sheep, and occasionally humans. Despite causing sustained infections, ORFV induces only a transient increase in pro-inflammatory signalling and the trafficking of innate immune cells within the skin seems to be impaired. An explanation for this tempered response to ORFV infection may lie in its expression of a homolog of the anti-inflammatory cytokine, interleukin (IL)-10. Using a murine model in which inflammation was induced by bacterial lipopolysaccharide, we examined the effects of the ORFV-IL-10 protein on immune cell trafficking to and from the skin. ORFV-IL-10 limited the recruitment of blood-derived Gr-1(int)/CD11b(int) monocytes, CD11c(+ve)/MHC-II(+ve) dendritic cells and c-kit(+ve)/FcεR1(+ve) mature mast cells into inflamed skin. ORFV-IL-10 also suppressed the activation of CD11c(+ve)/MHC-II(+ve) dendritic cells within the skin, reducing their trafficking to the draining lymph node. These findings suggest that expression of IL-10 by ORFV may contribute to the impaired trafficking of innate immune cells within infected skin. PMID:26732486

  13. 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

  14. Orf virus inhibits interferon stimulated gene expression and modulates the JAK/STAT signalling pathway.

    PubMed

    Harvey, Ryan; McCaughan, Catherine; Wise, Lyn M; Mercer, Andrew A; Fleming, Stephen B

    2015-10-01

    Interferons (IFNs) play a critical role as a first line of defence against viral infection. Activation of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway by IFNs leads to the production of IFN stimulated genes (ISGs) that block viral replication. The Parapoxvirus, Orf virus (ORFV) induces acute pustular skin lesions of sheep and goats and is transmissible to man. The virus replicates in keratinocytes that are the immune sentinels of skin. We investigated whether or not ORFV could block the expression of ISGs. The human gene GBP1 is stimulated exclusively by type II IFN while MxA is stimulated exclusively in response to type I IFNs. We found that GBP1 and MxA were strongly inhibited in ORFV infected HeLa cells stimulated with IFN-γ or IFN-α respectively. Furthermore we showed that ORFV inhibition of ISG expression was not affected by cells pretreated with adenosine N1-oxide (ANO), a molecule that inhibits poxvirus mRNA translation. This suggested that new viral gene synthesis was not required and that a virion structural protein was involved. We next investigated whether ORFV infection affected STAT1 phosphorylation in IFN-γ or IFN-α treated HeLa cells. We found that ORFV reduced the levels of phosphorylated STAT1 in a dose-dependent manner and was specific for Tyr701 but not Ser727. Treatment of cells with sodium vanadate suggested that a tyrosine phosphatase was responsible for dephosphorylating STAT1-p. ORFV encodes a factor, ORFV057, with homology to the vaccinia virus structural protein VH1 that impairs the JAK/STAT pathway by dephosphorylating STAT1. Our findings show that ORFV has the capability to block ISG expression and modulate the JAK/STAT signalling pathway. PMID:26113305

  15. Isolation and phylogenetic analysis of caprine Orf virus in Malaysia.

    PubMed

    Abdullah, Ashwaq Ahmed; Ismail, Muhammad Farid Bin; Balakrishnan, Krishnan Nair; Bala, Jamilu Abubakar; Hani, Homayoun; Abba, Yusuf; Awang Isa, Mohd Kamaruddin; Abdullah, Faez Firdaus Jesse; Arshad, Siti Suri; Nazariah, Zeenatul Allaudin; Abdullah, Rasedee; Mustapha, Noordin Mohamed; Mohd-Lila, Mohd-Azmi

    2015-12-01

    Orf virus is a DNA virus that causes contiguous ecthyma in goat and sheep. Infection of animals with this virus cause high mortality in young animals resulting in huge economic losses. In this study, we investigated an outbreak of Orf in a goat farm in Malaysia. Samples were collected from infected animals and viral isolation was done using both LT and MDCK cell lines. Molecular detection was done by conventional PCR for specific primers; B2L and F1L genes and phylogenetic analysis was done on the sequence data obtained. Cytopathic effects (CPE) were observed in both cell lines after 3 days of inoculation and were 50 % by the sixth day. PCR showed positive bands for both B2L and F1L genes and phylogenetic analysis showed that the Malaysian strain had close homology to the Chinese and Indian Orf virus isolates. This study gives more insight into the existing Orf viral strains in Malaysia and their relationship with other strains globally. PMID:26645035

  16. 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

  17. 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. PMID:26301926

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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

  4. 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. PMID:9514961

  5. Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System▿

    PubMed Central

    Zhang, Zhen; Rowe, Jenny; Wang, Weijia; Sommer, Marvin; Arvin, Ann; Moffat, Jennifer; Zhu, Hua

    2007-01-01

    To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo. PMID:17581997

  6. 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

  7. ORF157 from the Archaeal Virus Acidianus Filamentous Virus 1 Defines a New Class of Nuclease▿

    PubMed Central

    Goulet, Adeline; Pina, Mery; Redder, Peter; Prangishvili, David; Vera, Laura; Lichière, Julie; Leulliot, Nicolas; van Tilbeurgh, Herman; Ortiz-Lombardia, Miguel; Campanacci, Valérie; Cambillau, Christian

    2010-01-01

    Acidianus filamentous virus 1 (AFV1) (Lipothrixviridae) is an enveloped filamentous virus that was characterized from a crenarchaeal host. It infects Acidianus species that thrive in the acidic hot springs (>85°C and pH <3) of Yellowstone National Park, WY. The AFV1 20.8-kb, linear, double-stranded DNA genome encodes 40 putative open reading frames whose sequences generally show little similarity to other genes in the sequence databases. Because three-dimensional structures are more conserved than sequences and hence are more effective at revealing function, we set out to determine protein structures from putative AFV1 open reading frames (ORF). The crystal structure of ORF157 reveals an α+β protein with a novel fold that remotely resembles the nucleotidyltransferase topology. In vitro, AFV1-157 displays a nuclease activity on linear double-stranded DNA. Alanine substitution mutations demonstrated that E86 is essential to catalysis. AFV1-157 represents a novel class of nuclease, but its exact role in vivo remains to be determined. PMID:20200253

  8. Sheep-to-human transmission of Orf virus during Eid al-Adha religious practices, France.

    PubMed

    Nougairede, Antoine; Fossati, Christelle; Salez, Nicolas; Cohen-Bacrie, Stephan; Ninove, Laetitia; Michel, Fabrice; Aboukais, Samer; Buttner, Mathias; Zandotti, Christine; de Lamballerie, Xavier; Charrel, Remi N

    2013-01-01

    Five persons in France were infected with Orf virus after skin wounds were exposed to infected sheep tissues during Eid al-Adha, the Muslim Feast of Sacrifice. Infections were confirmed by electron microscopy, PCR, and sequence analysis. Prevention and control of this underdiagnosed disease can be achieved by educating physicians, slaughterhouse workers, and persons participating in Eid al-Adha. PMID:23260031

  9. Hepatitis E virus ORF1 encoded non structural protein-host protein interaction network.

    PubMed

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-02-01

    Hepatitis E virus ORF1 encoded non-structural polyprotein (nsP) consist of multiple domains, namely: Methyltransferase, Y-domain, Protease, X-domain, Helicase and RNA dependent RNA polymerase. We have attempted to identify human liver cell proteins that are interacting with HEV ORF1 encoded functional domains by using Y2H screening. A total of 155 protein-protein interactions between HEV-ORF1 and human proteins were identified. Comparative analysis of the HEV-ORF1-Human interaction network with reconstructed human interactome showed that the cellular proteins interacting with HEV-ORF1 are central and interconnected. Enrichment analysis of Gene Ontology and cellular pathways showed that the viral proteins preferentially interacted with the proteins of metabolism and energy generation along with host immune response and ubiquitin proteasomal pathways. The mTOR and focal adhesion pathways were also targeted by the virus. These interactions suggest that HEV probably utilizes important proteins in carbohydrate metabolism, energy generation and iron homoeostasis in the host cells during its establishment. PMID:26689634

  10. 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.

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

    PubMed

    Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana; de Rozières, Sohela; 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. PMID:17963812

  12. 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

  13. 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

  14. Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.

    PubMed

    Zhao, Qin; Sun, Ya-ni; Hu, Shou-bin; Wang, Xin-jie; Xiao, Yi-hong; Hsu, Walter H; Xiao, Shu-qi; Wang, Cheng-bao; Mu, Yang; Hiscox, Julian A; Zhou, En-Min

    2013-12-27

    Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection. PMID:24021883

  15. 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...

  16. Subcellular localization of grapevine red blotch-associated virus ORFs V2 and V3.

    PubMed

    Guo, Tai Wei; Vimalesvaran, Deluxsika; Thompson, Jeremy R; Perry, Keith L; Krenz, Björn

    2015-08-01

    Grapevine red blotch-associated virus is a recently discovered plant monopartite gemini-like virus found in North American grapevines. Leaf discoloration and a decrease in fruit quality are associated with its infection. Two of its six open reading frames (ORFs), V2 and V3, are of unknown function and share no obvious homology with plant or viral genes. Transient expression of these ORFs in fusion with the green fluorescent protein demonstrated that the V2 protein localizes in the nucleoplasm, Cajal bodies, and cytoplasm; and the V3 protein localizes in various unidentified subnuclear bodies. Additionally, the V2 protein is redirected to the nucleolus upon co-expression with the nucleolus and Cajal body-associated protein Fib2. PMID:26063598

  17. 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. PMID:26956473

  18. 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

  19. 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

  20. Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

    SciTech Connect

    Daniels, Candelaria C.; Rovnak, Joel; Quackenbush, Sandra L.

    2008-06-05

    Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assays and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKC{alpha}, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.

  1. Virological and clinico-pathological features of orf virus infection in experimentally infected rabbits and mice.

    PubMed

    Cargnelutti, J F; Masuda, E K; Martins, M; Diel, D G; Rock, D L; Weiblen, R; Flores, E F

    2011-01-01

    Many aspects of the biology of orf virus (ORFV) infection remain poorly understood and attempts to establish animal models have yielded conflicting and non-reproducible results. We herein describe the characterization of ORFV infection and disease in rabbits and mice. A protocol of intradermal inoculation was employed to inoculate 10(8.5)TCID₅₀/mL of ORFV strain IA-82 in the skin of ears, of the back and labial commissures. All inoculated rabbits presented a clinical course characterized by erythema, macules, papules/vesicles or pustules that eventually dried originating scabs. Local signs started around days 3 and 4 post-inoculation (pi) and lasted 3-10 days. Virus was recovered from lesions between days 2 and 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit milder clinical course occurred in 5/10 inoculated mice; virus was recovered from lesions from three animals. Inoculated lambs - used as controls - developed severe lesions of contagious ecthyma. VN tests performed at day 28pi failed to detect neutralizing antibodies in all inoculated animals. In contrast, convalescent rabbit sera were positive by ELISA at dilutions from 100 to 400. These results show that rabbits are susceptible to ORFV infection and thus may be used to study selected aspects of ORFV biology. PMID:20833245

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

    PubMed

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R; Lu, Shunwen

    2016-04-01

    Maize rayado fino virus (MRFV) possesses an open reading frame (ORF43) predicted to encode a 43 kDa protein (p43) 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 either prevent initiation from three potential AUG initiation codons near the 5'-end of ORF43 or prematurely terminate translation of ORF43. Inoculation of maize seed via vascular puncture inoculation (VPI) resulted in plants exhibiting symptoms typical of MRFV infection for all mutants tested. Furthermore, corn leafhoppers (Dalbulus maidis) transmitted the virus mutants to healthy plants at a frequency similar to that for wild-type MRFV-US. Viral RNA recovered from plants infected with mutants both prior to and after leafhopper transmission retained mutations blocking ORF43 expression. The results indicate that ORF43 of MRFV is dispensable for both systemic infection of maize and transmission by leafhoppers. PMID:26837893

  3. 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

  4. Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

    PubMed Central

    Tseng, Yeu-Yang; Lin, Fong-Yuan; Cheng, Sun-Fang; Chulakasian, Songkhla; Chou, Chia-Chi; Liu, Ya-Fen; Chang, Wei-Shan; Wong, Min-Liang

    2015-01-01

    ABSTRACT Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. PMID:25694596

  5. Evidence for the adaptive evolution of ORF5 gene of Porcine reproductive and respiratory syndrome virus isolated in China.

    PubMed

    Xu, Z; Chang, X; Xiao, S; Chen, H; Zhou, R

    2010-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene encoding an envelope glycoprotein involved in humoral immunity is the most variable protein-coding gene of PRRSV. The present study aimed to identify potential selective pressures acting on the ORF5 gene of PRRSV isolates of North American type prevalent in China. The non-synonymous to synonymous rate ratio ω (dN/dS) was employed as a measure of selective pressure at the codon level. An overall ω of 0.45 indicated negative (purifying) selection as the major driving force operating on the ORF5 gene during adaptation of the virus to swine. Determination of ω values for individual amino acids sites revealed 8 positively selected sites, most of them situated in the N-terminal ectodomain, indicating their potential role in the binding of virus to the cellular receptors. Further, 75 negatively selected sites were identified in the rest of molecule, probably as a result of functional or immunological constraints. Determination of potential N-glycosylation sites revealed 7 sites, four of which coincided with the positively selected ones. These results indicated that a specific adaptive evolution has operated on the ORF5 gene of Chinese PRRSV isolates. It is hoped that the disclosed adaptive sites might help identify a candidate antigenic epitope for the use in vaccine against this serious swine disease. PMID:21175251

  6. A Case of Orf Disease Complicated with Erythema Multiforme and Bullous Pemphigoid-Like Eruptions

    PubMed Central

    Alian, Shahriar; Ahangarkani, Fatemeh; Arabsheybani, Sara

    2015-01-01

    Parapoxvirus infection in sheep and goats is usually referred to as contagious pustular dermatitis/ecthyma, or orf, and the corresponding human infection is referred to as orf. In humans, after a brief incubation period of 3 to 5 days, lesions begin as pruritic erythematous macules and then rise to form papules, often with a target appearance. Lesions become nodular or vesicular, and orf lesions often ulcerate after 14 to 21 days. Erythema multiforme and bullous pemphigoid have been associated with parapoxvirus infections and they are rare complications of orf disease. In this case report, we presented a 36-year-old woman with history of contact with sheep, developing a typical orf lesion that is complicated with erythema multiforme and bullous pemphigoid-like eruptions. PMID:26294986

  7. A Case of Orf Disease Complicated with Erythema Multiforme and Bullous Pemphigoid-Like Eruptions.

    PubMed

    Alian, Shahriar; Ahangarkani, Fatemeh; Arabsheybani, Sara

    2015-01-01

    Parapoxvirus infection in sheep and goats is usually referred to as contagious pustular dermatitis/ecthyma, or orf, and the corresponding human infection is referred to as orf. In humans, after a brief incubation period of 3 to 5 days, lesions begin as pruritic erythematous macules and then rise to form papules, often with a target appearance. Lesions become nodular or vesicular, and orf lesions often ulcerate after 14 to 21 days. Erythema multiforme and bullous pemphigoid have been associated with parapoxvirus infections and they are rare complications of orf disease. In this case report, we presented a 36-year-old woman with history of contact with sheep, developing a typical orf lesion that is complicated with erythema multiforme and bullous pemphigoid-like eruptions. PMID:26294986

  8. Varicella-Zoster Virus ORF12 Protein Triggers Phosphorylation of ERK1/2 and Inhibits Apoptosis

    PubMed Central

    Liu, XueQiao; Li, Qingxue; Dowdell, Kennichi; Fischer, Elizabeth R.

    2012-01-01

    Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases involved in many cellular processes, including cell proliferation, differentiation, inflammation, and cell death. Activation of several MAPKs, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), results in stimulation of activator protein 1 (AP-1), which promotes gene transcription. Previous studies have demonstrated that varicella-zoster virus (VZV) infection activates ERK1/2, p38, and JNK to promote viral replication, but the underlying mechanism(s) is unclear. To identify viral proteins responsible for the activation of MAPK, we used a proteomic approach to screen viral proteins for AP-1 promoter activation by an AP-1–luciferase reporter assay. We found that VZV ORF12 protein, located in the tegument of virions, enhances AP-1 reporter activity. This effect of ORF12 protein was markedly inhibited by a MAPK/ERK kinase 1 and 2 (MEK1/2) inhibitor (U0126), partially blocked by a p38 inhibitor (SB202190), but not inhibited by a JNK inhibitor (SP600125). Expression of VZV ORF12 protein in cells resulted in phosphorylation of ERK1/2 and p38 but not JNK. Infection of cells with a VZV ORF12 deletion mutant resulted in reduced levels of phosphorylated ERK1/2 (p-ERK1/2) compared to infection with wild-type VZV. Furthermore, deletion of ORF12 rendered VZV-infected cells more susceptible to staurosporine-induced apoptosis. In conclusion, VZV ORF12 protein activates the AP-1 pathway by selectively triggering the phosphorylation of ERK1/2 and p38. Cells infected with a VZV ORF12 deletion mutant have reduced levels of p-ERK1/2 and are more susceptible to apoptosis than cells infected with wild-type VZV. PMID:22238304

  9. Enhancement of Interferon Induction by ORF3 Product of Hepatitis E Virus

    PubMed Central

    Nan, Yuchen; Ma, Zexu; Wang, Rong; Yu, Ying; Kannan, Harilakshmi; Fredericksen, Brenda

    2014-01-01

    ABSTRACT Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion. IMPORTANCE Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA

  10. 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. PMID:24189622

  11. 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

  12. The ORF61 Protein Encoded by Simian Varicella Virus and Varicella-Zoster Virus Inhibits NF-κB Signaling by Interfering with IκBα Degradation

    PubMed Central

    Whitmer, Travis; Malouli, Daniel; Uebelhoer, Luke S.; DeFilippis, Victor R.

    2015-01-01

    ABSTRACT Varicella-zoster virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by postherpetic neuralgia. Innate immunity mediated by interferon and proinflammatory cytokines represents the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways, including the central transcription factor NF-κB. However, the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV) infection of rhesus macaques recapitulates key aspects of VZV pathogenesis, and this model thus permits examination of the role of immune evasion mechanisms in vivo. Here, we compare SVV and VZV with respect to interference with NF-κB activation. We demonstrate that both viruses prevent ubiquitination of the NF-κB inhibitor IκBα, whereas SVV additionally prevents IκBα phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IκBα ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with β-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IκBα. This interaction seems to inactivate SCF-mediated protein degradation in general, since the unrelated β-TrCP target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NF-κB pathway, since SVV with ORF61 deleted still prevented IκBα phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with tumor necrosis factor alpha (TNF-α)-induced NF-κB activation at multiple levels, which is consistent with the importance of these countermechanisms for varicella virus infection. IMPORTANCE The role of innate immunity during the establishment of primary infection, latency, and reactivation by varicella-zoster virus (VZV) is incompletely understood. Since

  13. 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

  14. 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. PMID:26801789

  15. Quantification of Maize Fine Streak Virus Genomic and Positive-sense RNAs in Infected Maize Reveals High Level Accumulation of ORF 3 and 4 MFSV Transcripts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantification of Maize fine streak virus genomic and positive-sense RNAs in infected maize reveals high level accumulation of ORF 3 and 4 MFSV transcripts. We improved methods to analyze RNA produced by Maize fine streak virus (MVSF) within infected maize tissue using real-time RT-qPCR. We designe...

  16. 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

  17. 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

  18. 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. PMID:20861245

  19. Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

    PubMed

    Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

    2013-01-01

    The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a

  20. 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

  1. 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

    2016-01-01

    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 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. PMID:27113483

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

    PubMed Central

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

    2015-01-01

    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

  3. Development and comparative evaluation of loop mediated isothermal amplification (LAMP) assay for simple visual detection of orf virus in sheep and goats.

    PubMed

    Venkatesan, G; Bhanuprakash, V; Balamurugan, V

    2015-06-01

    A loop-mediated isothermal amplification (LAMP) assay targeting DNA Pol gene was optimized and evaluated for the rapid detection of orf virus in clinical samples. The LAMP assay was found to be specific and sensitive. The detection rate of LAMP (89.3%) was better than PCR (67.9%) and comparable to real-time PCR (91.1%) in clinical samples by gel electrophoresis and visual detection methods. This LAMP assay is simple and does not rely upon any special equipment and could be employed in clinical diagnosis and epidemiological survey of orf infection. PMID:25828693

  4. Immune gene expression in swine macrophages expressing the Torque Teno Sus Virus1 (TTSuV1) ORF-1 and 2 proteins.

    PubMed

    Singh, Pankaj; Ramamoorthy, Sheela

    2016-07-15

    Torque Teno viruses (TTVs) are small DNA viruses which are ubiquitous in nature. Recent reports indicate that swine torque teno viruses (TTSuVs) can act as primary pathogens or play a role in exacerbating co-infections. However, very little is known about the TTSuV host-viral interaction or how they so successfully establish chronic infections in the host. To determine whether the major viral proteins can modulate host immunity, recombinant TTSuV1 ORF1 and 2 proteins were expressed in a swine macrophage cell line (3D4/31). The differential expression of a panel of innate, adaptive, regulatory and inflammatory immune genes was studied by quantitative PCR; using cDNA samples collected at 6, 12, 24 and 48h post-transfection. The ORF1 protein induced an early anti-viral response. However, at 6h post-transfection it also upregulated IL-10, PD-1 and SOCS-1, the suppressors of T cell mediated immunity. An ensuing diminishment of the early protective response was noted. The TTSuV1 ORF2 protein suppressed IFN-β and IL-13 responses but did not significantly influence anti-viral immunity otherwise. These findings indicate that the TTSuV1 ORF1 protein plays a significant but dual role in viral immunity. PMID:27059616

  5. ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) is a target of diversifying selection as infection progresses from acute infection to virus rebound.

    PubMed

    Chen, Nanhua; Trible, Benjamin R; Kerrigan, Maureen A; Tian, Kegong; Rowland, Raymond R R

    2016-06-01

    Genetic variation in both structural and nonstructural genes is a key factor in the capacity of porcine reproductive and respiratory syndrome virus (PRRSV) to evade host defenses and maintain within animals, farms and metapopulations. However, the exact mechanisms by which genetic variation contribute to immune evasion remain unclear. In a study to understand the role of host genetics in disease resistance, a population of pigs were experimentally infected with a type 2 PRRSV isolate. Four pigs that showed virus rebound at 42days post-infection (dpi) were analyzed by 454 sequencing to characterize the rebound quasispecies. Deep sequencing of variable regions in nsp1, nsp2, ORF3 and ORF5 showed the largest number of nucleotide substitutions at day 28 compared to days 4 and 42 post-infection. Differences were also found in genetic variations when comparing tonsil versus serum. The results of dN/dS ratios showed that the same regions evolved under negative selection. However, eight amino acid sites were identified as possessing significant levels of positive selection, including A27V and N32S substitutions in the GP5 ectodomain region. These changes may alter GP5 peptide signal sequence processing and N-glycosylation, respectively. The results indicate that the greatest genetic diversity occurs during the transition between acute and rebound stages of infection, and the introduction of mutations that may result in a gain of fitness provides a potential mechanism for persistence. PMID:26961593

  6. 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

  7. 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

  8. 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

  9. 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. PMID:27170377

  10. Differentiation of Varicella-Zoster Virus ORF47 Protein Kinase and IE62 Protein Binding Domains and Their Contributions to Replication in Human Skin Xenografts in the SCID-hu Mouse

    PubMed Central

    Besser, Jaya; Sommer, Marvin H.; Zerboni, Leigh; Bagowski, Christoph P.; Ito, Hideki; Moffat, Jennifer; Ku, Chia-Chi; Arvin, Ann M.

    2003-01-01

    To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo. PMID:12719588

  11. Walleye Dermal Sarcoma Virus: OrfA N-Terminal End Inhibits the Activity of a Reporter Gene Directed by Eukaryotic Promoters and Has a Negative Effect on the Growth of Fish and Mammalian Cells

    PubMed Central

    Zhang, Z.; Martineau, D.

    1999-01-01

    Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis. The WDSV genome contains three short open reading frames designated orfA, orfB, and orfC in addition to the viral structural genes, gag, pol, and env. orfA and orfB transcripts are detected in tumors by reverse transcription-PCR. Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain. We report that expression of the accessory gene orfA inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters. In addition, stable transfection with the wild-type orfA generated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector. An orfA mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects. PMID:10482648

  12. Understanding the role of ORF-C gene in the pathogenicity of infectious laryngotracheitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a very serious and widespread respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Conventional attenuated ILT vaccines, obtained by continuous passages in chicken embryos and tissue culture, had been the main tools utilized by th...

  13. 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

  14. 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

  15. Protein encoded by ORF093 is an effective vaccine candidate for infectious spleen and kidney necrosis virus in Chinese perch Siniperca chuatsi.

    PubMed

    Li, Ningqiu; Fu, Xiaozhe; Guo, Huizhi; Lin, Qiang; Liu, Lihui; Zhang, Defeng; Fang, Xiang; Wu, Shuqin

    2015-01-01

    Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease causing high mortality and economic losses in Chinese perch, Siniperca chuatsi in China. Little information about the antigenicity of ISKNV proteins is available. In this study the ORF093 gene of ISKNV was cloned into the prokaryotic expression vector pET32a(+) and eukaryotic expression vector pcDNA3.1(+), and designated as pET-093 and pcDNA-093, respectively. A recombinant 51-kDa protein was detected in Escherichia coli BL21 (harboring pET-093) after IPTG inducement. Polyclonal antibodies were raised in rabbits against the purified ORF093 protein and the reaction of the antibodies was confirmed by western blotting using the purified recombinant protein. Expression of the pcDNA-093 in muscle tissue of vaccinated fish was confirmed by western blotting. The protection efficacy of ORF093 was investigated and results showed that cumulative mortality of Chinese perch was significant differences between immune groups and control (P<0.05) after ISKNV challenge, and the RPS value of 093 recombinant protein, pcDNA093 and pcDNA093+MCP was 47%, 50% and 57%. The results suggested that ORF093 is an effective vaccine candidate for ISKNV and it can be used in the control of ISKNV disease in Chinese perch. PMID:25462463

  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. 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.

  19. Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

    PubMed Central

    Yang, Ming; Wang, Shuo; Yue, Xiu-Li

    2014-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCE To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132

  20. AIDS Vaccination Studies with an Ex Vivo Feline Immunodeficiency Virus Model: Analysis of the Accessory ORF-A Protein and DNA as Protective Immunogens

    PubMed Central

    Pistello, Mauro; Bonci, Francesca; Flynn, J. Norman; Mazzetti, Paola; Isola, Patrizia; Zabogli, Elisa; Camerini, Valentina; Matteucci, Donatella; Freer, Giulia; Pelosi, Paolo; Bendinelli, Mauro

    2006-01-01

    Determining which antigen must be included in AIDS vaccines to confer maximum protection is of utmost importance. In primate models, vaccines consisting of or including accessory viral proteins have yielded conflicting results. We investigated the protective potential of the accessory protein ORF-A of feline immunodeficiency virus (FIV) in cats. All three immunization strategies used (protein alone in alum adjuvant, DNA alone, or DNA prime-protein boost) clearly generated detectable immune responses. Upon challenge with ex vivo homologous FIV, ORF-A-immunized cats showed distinct enhancement of acute-phase infection relative to mock-immunized animals given alum or empty vector DNA. This effect was tentatively attributed to increased expression of the FIV receptor CD134 that was observed in the immunized cats. However, at subsequent sampling points that were continued for up to 10 months postchallenge, the average plasma viral loads of the ORF-A-immunized animals were slightly but consistently reduced relative to those of the control animals. In addition, CD4+ T lymphocytes in the circulation system declined more slowly in immunized animals than in control animals. These findings support the contention that immunization with lentiviral accessory proteins can improve the host's ability to control virus replication and slow down disease progression but also draw attention to the fact that even simple immunogens that eventually contribute to protective activity can transiently exacerbate subsequent lentiviral infections. PMID:16940498

  1. Early protein ORF086 is an effective vaccine candidate for infectious spleen and kidney necrosis virus in mandarin fish Siniperca chuatsi.

    PubMed

    Fu, Xiaozhe; Li, Ningqiu; Lin, Qiang; Guo, Huizhi; Liu, Lihui; Huang, Zhibin; Wu, Shuqin

    2015-10-01

    Infectious spleen and kidney necrosis virus (ISKNV) has caused significant loss in the Mandarin fish (Siniperca chuatsi) aquaculture industry. Vaccination is an important measure to prevent fatal ISKNV infection. In this study, the ORF086 gene encoding an early protein helicase of ISKNV was cloned into the prokaryotic pET32a (+) and eukaryotic pcDNA3.1 (+) expression vectors and designated as pET086 and pcDNA086, respectively. A recombinant 36 kDa protein was detected in Escherichia coli BL21 (harboring pET086) after isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. Polyclonal antibodies against the purified ORF086 protein were raised in rabbits. The antibody reaction and the pcDNA086 expression in muscle tissues of vaccinated fish were confirmed using Western blot analysis. The protective efficacy of ORF086 was also investigated. The cumulative mortality rates of Mandarin fish were significantly different between immune and control groups (P < 0.05) after ISKNV challenge. The relative percentage survival (RPS) values of the recombinant ORF086 protein emulsified with ISA763A adjuvant and pcDNA086 added with QCDC adjuvant were 73% and 63%, respectively. Transcriptional analysis of non-specific and specific immune related genes revealed that the expression levels of IRF-7, IRAK1, Mx, Viperin, and IgM were strongly up-regulated in the vaccinated groups post-immunization. In particular, the expression levels in the QCDC + pcDNA086 group was higher than those in the control groups (P < 0.05). These results indicated that the early protein ORF086 could be an effective antigen candidate for controlling ISKNV disease in Mandarin fish. PMID:26099219

  2. Tracing the genetic history of porcine reproductive and respiratory syndrome viruses derived from the complete ORF 5-7 sequences: a Bayesian coalescent approach.

    PubMed

    Yoon, Sook Hee; Kim, Hyekwon; Park, Bongkyun; Kim, Heebal

    2012-11-01

    To trace the genetic history of porcine reproductive and respiratory syndrome virus (PRRSV), we determined the complete sequences of ORFs 5 to 7 of four PRRSV isolates. These sequences were analyzed together with published sequences from 146 isolates from various parts of the world using a Bayesian coalescent approach as well as Bayesian inference and maximum-likelihood methods. All of the European-type (EU-type) viruses were classified into one of two groups or unclassified (4 isolates), while all North American-type (NA-type) viruses belonged to one of three major groups or were unclassified (5 isolates). Within each genotype, no apparent periodic and/or geographic influence on the evolution of PRRSVs was observed. The evolutionary rate of PRRSV isolates was estimated to be 1.55 × 10(-3) substitutions/site/year, and the time of the most recent common ancestor (TMRCA) was 491.2 years ago. Here, the TMRCA for the EU- and NA-type viruses was 58.7 and 62.6 years ago, respectively. A Bayesian skyline plot revealed that the viruses evolved at an almost constant population size until the late 1970s, when they experienced a population expansion that continued until the late 1980s. The population size then remained constant again until the early 2000s, when a rapid, sharp decline in the effective number of infections occurred. PMID:22825696

  3. 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. PMID:25725149

  4. 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. PMID:10573162

  5. 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

  6. 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

  7. Orf-I and Orf-II-Encoded Proteins in HTLV-1 Infection and Persistence

    PubMed Central

    Edwards, Dustin; Fenizia, Claudio; Gold, Heather; de Castro-Amarante, Maria Fernanda; Buchmann, Cody; Pise-Masison, Cynthia A.; Franchini, Genoveffa

    2011-01-01

    The 3′ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. Here, we review current knowledge of HTLV-1 orf-I and orf-II protein products. Singly spliced mRNA from orf-I encodes p12, which can be proteolytically cleaved to generate p8, while differential splicing of mRNA from orf-II results in production of p13 and p30. These proteins have been demonstrated to modulate transcription, apoptosis, host cell activation and proliferation, virus infectivity and transmission, and host immune responses. Though these proteins are not essential for virus replication in vitro, p8, p12, p13, and p30 have an important role in the establishment and maintenance of HTLV-1 infection in vivo. PMID:21994758

  8. Rescue of Wild-type Mumps Virus from a Strain Associated with Recent Outbreaks Helps to Define the Role of the SH ORF in the Pathogenesis of Mumps Virus

    PubMed Central

    Xu, Pei; Li, Zhuo; Sun, Dengyun; Lin, Yuan; Wu, Jianguo; Rota, Paul A.; He, Biao

    2011-01-01

    Mumps virus (MuV) causes acute infections in humans. In recent years, MuV has caused epidemics among highly vaccinated populations. The largest outbreak in the U.S. in the past 20 years occurred in 2005–2006 with over reported 5,000 cases which the majority of the cases was in vaccinated young adults. We sequenced the complete genome of a representative strain from the epidemic (MuV-IA). MuV-IA is a member of genotype G, the same genotype of MuV that was associated with the outbreak in the UK in 2004–2005. We constructed a reverse genetics system for MuV-IA (rMuV-IA), and rescued a virus lacking the open reading frame (ORF) of the SH gene (rMuVΔSH). rMuVΔSH infection in L929 cells induced increased NF-κB activation, TNF-α production and apoptosis compared to rMuV-IA. rMuVΔSH was attenuated in an animal model. These results indicated that the SH ORF of MuV plays a significant role in interfering with TNF-α signaling and viral pathogenesis during virus infection. PMID:21676427

  9. 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

  10. 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

  11. 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

  12. ORF3 of Hepatitis E Virus Inhibits the Expression of Proinflammatory Cytokines and Chemotactic Factors in LPS-Stimulated Human PMA-THP1 Cells by Inhibiting NF-κB Pathway.

    PubMed

    Lei, Qingsong; Li, Lin; Cai, Jia; Huang, Wenxiang; Qin, Bo; Zhang, Shujun

    2016-03-01

    Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis. It is noteworthy that HEV can develop chronic infection and even lead to liver cirrhosis; however, the mechanism has not been revealed. In this study, the ELISA assay was used to detect protein levels, and we found that HEV open reading frame 3 (ORF3) protein inhibited the expression of proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, IL-6, IL-8, IL-12p40, and IL-18) and chemotactic factors (nitric oxide [NO], interferon-inducible protein-10 (IP-10), macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein-1 (MCP-1), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF)] in lipopolysaccharide (LPS)-stimulated human PMA-THP1 cells. Further study showed that mRNA and protein levels of pattern recognition receptors (PRRs), such as Toll-like receptor 4 (TLR4), TNF receptor-associated factor 6 (TRAF6), and nucleotide-binding oligomerization domain containing 2 (NOD2), decreased after infection of pLL3.7-ORF3 (pORF3); moreover, the inhibition produced corresponding upregulation of IκBα and downregulation of phosphorylated IκB kinase IKKɛ (p-IKKɛ) and phosphorylated nuclear factor (NF)-κB (p-NF-κB), but little variation was found in the concentration of IKKɛ and NF-κB. Taken together, our results demonstrated that HEV ORF3 attenuated LPS-induced cytokine production and chemotactic factors, predominantly by inhibiting various PRRs-mediated NF-κB signaling pathways. The anti-inflammatory properties might be of great importance to clarify the role and mechanism of macrophages in chronic HEV infection and cirrhosis. PMID:26771290

  13. Inactivated Parapoxvirus ovis induces a transient increase in the expression of proinflammatory, Th1-related, and autoregulatory cytokines in mice

    PubMed Central

    Anziliero, D.; Weiblen, R.; Kreutz, L.C.; Spilki, F.; Flores, E.F.

    2014-01-01

    The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines. PMID:24519126

  14. 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.

  15. Analysis of ORF5 and Full-Length Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Isolates of Genotypes 1 and 2 Retrieved Worldwide Provides Evidence that Recombination Is a Common Phenomenon and May Produce Mosaic Isolates

    PubMed Central

    Martín-Valls, G. E.; Kvisgaard, L. K.; Tello, M.; Darwich, L.; Cortey, M.; Burgara-Estrella, A. J.; Hernández, J.; Larsen, L. E.

    2014-01-01

    ABSTRACT Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) isolates by examining previously published data sets of ORF5 sequences (genotypes 1 and 2) obtained worldwide. We then examined full-length genome sequences in order to determine potential recombination breakpoints along the viral genome. For ORF5, 11 sets of genotype 1 sequences from different geographical areas, including 2 Asian, 1 American, and 7 European regions, and three sets of genotype 2, including sets from China, Mexico, and the United States, were analyzed separately. Potential recombination breakpoints were detected in 10/11 genotype 1 sets, including 9 cases in which the clustering of at least one isolate was different before and after the breakpoints. In genotype 2, potential breakpoints and different tree clustering of at least one strain before and after the breakpoint were observed in 2 out of 3 sets. The results indicated that most of the ORF5 data sets contained at least one recombinant sequence. When the full-length genome sequences were examined, both genotype 1 and 2 sets presented breakpoints (10 and 9, respectively), resulting in significantly different topologies before and after the breakpoints. Mosaic genomes were detected in genotype 1 sequences. These results may have significant implications for the understanding of the molecular epidemiology of PRRSV. IMPORTANCE PRRSV is one of the most important viruses affecting swine production worldwide, causing big economic losses and sanitary problems. One of the key questions on PRRSV arises from its genetic diversity, which is thought to have a direct impact on immunobiology, epidemiology, diagnosis, and vaccine efficacy. One of the causes of this genetic diversity is

  16. Detection and quantification of parapoxvirus DNA by use of a quantitative real-time polymerase chain reaction assay in calves without clinical signs of parapoxvirus infection.

    PubMed

    Yaegashi, Gakuji; Fukunari, Kazuhiro; Oyama, Takayuki; Murakami, Ryu-Koh; Inoshima, Yasuo

    2016-04-01

    OBJECTIVE To investigate the presence of parapoxvirus (PPV) in cattle without clinical signs of infection and in farm environments of PPV-infected cattle. ANIMALS 28 calves without clinical signs of PPV infection on 2 farms and 11 clinically affected calves on 6 farms. PROCEDURES 164 oral swab samples were collected at regular intervals from 28 calves without clinical signs of PPV infection, and 11 swab samples were collected from 11 clinically affected calves. Viral DNA load was quantified by use of a PPV-specific quantitative real-time PCR (qRT-PCR) assay. RESULTS Of 28 calves without clinical signs of PPV infection, 12 had positive results for PPV DNA by use of the qRT-PCR assay. Viral DNA was detected continuously over a period of 2 to 5 months from 9 of these 12 calves, particularly from calves with dermatomycosis or respiratory tract disease. The PPV DNA loads in 32 oral swab samples from these 12 calves were significantly lower (median, 3.2 copies/mg) than those in samples collected from the 11 clinically affected calves (median, 3.2 × 10(4) copies/mg). Moreover, PPV DNA was detected in the residual feed and drinking water on both farms that housed the calves without clinical signs of PPV infection. CONCLUSIONS AND CLINICAL RELEVANCE PPV in cattle without clinical signs of infection and in the environments of these cattle may represent sources of PPV transmission to susceptible cattle. IMPACT FOR HUMAN MEDICINE Humans should wear gloves to prevent zoonotic disease transmission when handling cattle with or without clinical signs of PPV infection. PMID:27027837

  17. 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

  18. Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008.

    PubMed

    Zhou, Yan-Jun; Yu, Hai; Tian, Zhi-Jun; Li, Guo-Xin; Hao, Xiao-Fang; Yan, Li-Ping; Peng, Jin-Mei; An, Tong-Qing; Xu, Ao-Tian; Wang, Ya-Xin; Wei, Tian-Chao; Zhang, Shan-Rui; Cai, Xue-Hui; Feng, Li; Li, Xi; Zhang, Gui-Hong; Zhou, Lun-Jiang; Tong, Guang-Zhi

    2009-09-01

    Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5-100% nucleotide and 98.3-100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II-VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I-III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China. PMID:19406176

  19. 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...

  20. Attenuation, transmission, and immunogenicity of an ORF-C gene deleted strain of infectious laryngotracheitis virus (ILTV) in specific pathogen free chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a very serious and widespread respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Conventional attenuated ILT vaccines, obtained by continuous passages in chicken embryos and tissue culture, had been the main tools utilized by th...

  1. 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

  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. PMID:24771532

  3. Bombyx mori nucleopolyhedrovirus orf8 encodes a nucleic acid binding protein that colocalizes with IE1 during infection.

    PubMed

    Imai, N; Kurihara, M; Matsumoto, S; Kang, W-K

    2004-08-01

    This report describes the characterization of the Bombyx mori nucleopolyhedrovirus (BmNPV) orf8 gene. Immunoblot analyses demonstrated that orf8 was expressed as an early gene. The ORF8 protein accumulated in the nucleus, and was maintained at relatively constant levels from 4 to 24 h postinfection. Immunoblot analysis failed to detect ORF8 protein associated with budded virus and occlusion derived virus. In addition, immunohistochemical analysis by confocal microscopy showed that ORF8 protein colocalized with IE1 to specific nuclear foci throughout infection. To further examine the function of ORF8, a reporter gene was inserted into the orf8 reading frame. One orf8 disruption mutant (BmD8), which expressed the N-terminal half of ORF8, was isolated. However, it was not possible to isolate a null mutant, suggesting that orf8 may have an important role during viral infection. Single-step growth curves showed that BV production was reduced in BmD8 infected cells. Biochemical analyses indicated that ORF8 bound to nucleic acids. Together, these results suggest that BmNPV ORF8 may be involved in viral DNA replication and/or transcription. PMID:15290382

  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. [The expression of porcine circovirus type 2 ORF2 gene in insect cells and its character].

    PubMed

    Fan, Hui-Ying; Chen, Huan-Chun; Tong, Tie-Zhu; Ju, Chun-Mei; Lu, Jian-Qiang; Huang, Hong-Liang

    2005-11-01

    To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter. PMID:16468356

  6. Canine herpesvirus ORF2 is a membrane protein modified by N-linked glycosylation.

    PubMed

    Nishikawa, Yoshifumi; Kimura, Michiko; Xuan, Xuenan; Makala, Levi; Nagasawa, Hideyuki; Mikami, Takeshi; Otsuka, Haruki

    2002-07-01

    Canine herpesvirus (CHV) ORF2, located downstream of the glycoprotein C (gC) gene, has homologues with some of the alphaherpesviruses. To characterize CHV OFR2, a recombinant CHV carrying a LacZ gene in the ORF2 locus, and recombinant vaccinia virus expressing ORF2 protein were constructed. Northern blot analysis revealed ORF2 and a gamma2 class late gene, and its protein product was detectable in CHV-infected cells reacted with ORF2 protein antiserum. Tunicamycin and N-glycosidase F treatment revealed that the ORF2 protein was modified by N-linked glycosylation. Fractionation and immune fluorescence analyses of the CHV-infected cells showed the ORF2 as a membrane protein transportable to the surface of infected cells. In vitro, the ORF2 protein did not affect viral replication and cell-to-cell viral spreading. Present findings represent the first evidence pointing to the CHV ORF2 as a membrane protein modified by an N-linked glycosylation. PMID:12135784

  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. Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes.

    PubMed

    Mahé, D; Blanchard, P; Truong, C; Arnauld, C; Le Cann, P; Cariolet, R; Madec, F; Albina, E; Jestin, A

    2000-07-01

    Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated. PMID:10859388

  9. Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium.

    PubMed

    Pohlscheidt, M; Langer, U; Minuth, T; Bödeker, B; Apeler, H; Hörlein, H-D; Paulsen, D; Rübsamen-Waigmann, H; Henzler, H-J; Reichl, U

    2008-03-17

    For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10 L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50 L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called "Volume-Expanded-Fed" Batch with cell densities up to 6-7e06 cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved. PMID:18295380

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

    PubMed Central

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

    2015-01-01

    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

  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. 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

  13. 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.

  14. Stability of the WSSV ORF94 VNTR genotype marker during passage in marine shrimp, freshwater crayfish and freshwater prawns.

    PubMed

    Gudkovs, Nicholas; Murwantoko, Murwantoko; Walker, Peter J

    2014-10-16

    The white spot syndrome virus (WSSV) genome contains 3 variable number tandem repeat (VNTR) regions, located in open reading frame (ORF) 75, ORF94 and ORF125, which have been employed for molecular epizootiological studies. A previous report suggested that the ORF 94 VNTR is highly unstable, varying in the number of tandem repeats during single passages from shrimp to other crustaceans. As such rapid variations would have profound implications for the interpretation of molecular epizootiological data, we re-examined the stability of the ORF94 VNTR. Two WSSV isolates with different ORF94 VNTR genotypes (TRS5 and TRS7) were obtained from disease outbreaks in farmed black tiger shrimp Penaeus monodon in Indonesia. High titre stocks of each virus were produced by injection in specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei with filtered infected tissue extracts, and the genotypes were confirmed. Each stock (macerated tissue) was then used to feed SPF Pacific white shrimp, freshwater crayfish (Cherax sp.) and freshwater prawns Macrobrachium rosenbergii through 3 successive passages involving alternative hosts at each level. Taqman real-time PCR was conducted on samples from each group to confirm infection and quantify viral genetic loads. ORF94 VNTR genotype analysis conducted on samples from each of the 43 passage groups indicated no variations in the VNTR number in either genotype TRS5 or genotype TRS7. This finding is contrary to the previous report and suggests that ORF94 VNTR are stable during multiple passages in these crustaceans. PMID:25320037

  15. 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...

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

    PubMed Central

    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

  17. ORF8-Related Genetic Evidence for Chinese Horseshoe Bats as the Source of Human Severe Acute Respiratory Syndrome Coronavirus.

    PubMed

    Wu, Zhiqiang; Yang, Li; Ren, Xianwen; Zhang, Junpeng; Yang, Fan; Zhang, Shuyi; Jin, Qi

    2016-02-15

    Several lineage B betacoronaviruses termed severe acute respiratory syndrome (SARS)-like CoVs (SL-CoVs) were identified from Rhinolophus bats in China. These viruses are characterized by a set of unique accessory open reading frames (ORFs) that are located between the M and N genes. Among unique accessory ORFs, ORF8 is most hypervariable. In this study, the ORF8s of all SL-CoVs were classified into 3 types, and, for the first time, it was found that very few SL-CoVs from Rhinolophus sinicus have ORF8s that are identical to that of human SARS-CoV. This finding provides new genetic evidence for Chinese horseshoe bats as the source of human SARS-CoV. PMID:26433221

  18. Complete nucleotide sequence of Rose yellow leaf virus, a new member of the family Tombusviridae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome of the Rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides containing 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 a 87 kDa (p87) protein t...

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus.

    PubMed

    Meier, Christoph; Aricescu, A Radu; Assenberg, Rene; Aplin, Robin T; Gilbert, Robert J C; Grimes, Jonathan M; Stuart, David I

    2006-07-01

    To achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like beta structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds. PMID:16843897

  5. Comparative in vivo analysis of recombinant type II feline coronaviruses with truncated and completed ORF3 region.

    PubMed

    Bálint, Ádám; Farsang, Attila; Zádori, Zoltán; Belák, Sándor

    2014-01-01

    Our previous in vitro comparative study on a feline coronavirus (FCoV) pair, differing only in the intactness of their ORF3abc regions, showed that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II feline infectious peritonitis virus (FIPV). In the present study, we describe a challenge experiment with the same recombinant FCoVs in order to gain data on the in vivo characteristics on these viruses. While parent virus FIPV DF-2 developed feline infectious peritonitis in all the infected cats, its recombinant virus PBFIPV-DF-2, differing only in seven nucleotides, proved to be surprisingly low virulent, although caused an acute febrile episode similarly to the original FIPV DF-2. PBFIPV-DF-2 infection induced significantly lower virus neutralization titers than its parent virus, and lacked the second phase of viremia and development of fatal course of the disease. The recombinant PBFIPV-DF-2-R3i with completed ORF3abc gained biological properties that differentiate between the feline enteric coronavirus (FECV) and FIPV biotypes such as intensive replication in the gut, absence of viremia and weak or no serological response. Using reverse genetic approaches our study is the first experimental proof that ORF3abc is indeed responsible for the restriction of FECV replication to the intestine in vivo. PMID:24586385

  6. 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. PMID:25218850

  7. 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...

  8. Computer analysis between nucleotide and amino acid sequences of bean golden mosaic virus and those of maize streak, wheat dwarf, chloris striate mosaic, and beet curly top viruses.

    PubMed

    Ikegami, M

    1989-01-01

    Bean golden mosaic virus (BGMV) DNA 1 and 2 have little sequence homology with maize streak virus (MSV), wheat dwarf virus (WDV), and chloris striate mosaic virus (CSMV) DNAs. BGMV DNA 1 and beet curly top virus (BCTV) DNA are closely related, whereas BGMV DNA 2 and BCTV DNA are not related. Direct amino acid homologies of predicted proteins between BGMV ORFs and MSV ORFs, WDV ORFs or CSMV ORFs were 40-50%. BGMV 1L1 and BCTV L1, and BGMV IL3 and BCTV L4 were highly conserved. The sequence TAATATTAC was detected in the loops of hairpin structures of 5 gemini-viruses. PMID:2615677

  9. 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.

  10. Genome-wide mutagenesis reveals that ORF7 is a novel VZV skin-tropic factor.

    PubMed

    Zhang, Zhen; Selariu, Anca; Warden, Charles; Huang, Grace; Huang, Ying; Zaccheus, Oluleke; Cheng, Tong; Xia, Ningshao; Zhu, Hua

    2010-01-01

    The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles. PMID:20617166

  11. 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

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

    PubMed

    Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

    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

  13. [Construction and identification of a recombinant PRRSV expressing ORF2 of porcine circovirus type 2].

    PubMed

    Zhang, Tingjie; Liu, Xing; Sun, Tao; Zhu, Xuejiao; Fan, Baochao; Bai, Juan; Jiang, Ping

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are very two important pathogens that have coursed huge economic losses in swine production in worldwide. In this study,a vector pCMV-TJM containing the full-length cDNA clone of PRRSV attenuated strain TJM-F92 was firstly constructed by PCR method. Then a gene sequence containing Afl II/Mlu I e restriction enzyme sites and a transcription regulatory sequence for ORF6 (TRS6) was inserted be- tween ORF7 and 3'UTR, yielding a expression vector pCMV-TJM-TRS. Subsequently, a plasmid pCMV-TJM-Cap was constructed by cloning of PCV2 ORF2 gene into the unique sites Afl II /Mlu I of pCMV- TJM-TRS plasmid DNA. Then three recombinant PRRSV, rTJM, rTJM/TRS and rTJM/Cap, were rescued by transfection of pCMV-TJM, pCMV-TJM-TRS and pCMV-TJM-Cap into Marc-145 cells, respectively,and confirmed by the genome sequence, restriction enzyme digestion, Western Blot and IFA. They all had the molecular markers which was different from the parent virus. The growth characteristics of the rescued viruses were similar to that of parent virus. rTJM/Cap could also express efficiently PCV2 Cap protein in Marc-145 cells. At passage 8, it still had PCV2 ORF2 gene which examined by RT-PCR. It indicated that the full-length cDNA clone of PRRSV attenuated strain TJM-F92 and recombinant PRRSV rTJM/Cap expressing PCV2 Cap protein were successfully constructed. It made an important foundation for studying on the pathogenic mechanisms of PRRSV and PRRSV-PCV2 vaccine in the future. PMID:25997333

  14. Vaccination of mice with ORF 5 plasmid DNA of PRRSV; enhanced effects by co-immunizing with porcine IL-15

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The open reading frame (ORF) 5 of porcine reproductive and respiratory syndrome virus (PRRSV) encodes a major envelope glycoprotein designated GP5. The GP5 protein is a candidate for developing vaccines against PRRSV infection. In this study, recombinant plasmids bearing the PRRSV GP5 gene or the po...

  15. 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

  16. 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

  17. 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

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  19. 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

  20. 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

  1. Upstream ORFs are prevalent translational repressors in vertebrates.

    PubMed

    Johnstone, Timothy G; Bazzini, Ariel A; Giraldez, Antonio J

    2016-04-01

    Regulation of gene expression is fundamental in establishing cellular diversity and a target of natural selection. UntranslatedmRNAregions (UTRs) are key mediators of post-transcriptional regulation. Previous studies have predicted thousands ofORFs in 5'UTRs, the vast majority of which have unknown function. Here, we present a systematic analysis of the translation and function of upstream open reading frames (uORFs) across vertebrates. Using high-resolution ribosome footprinting, we find that (i)uORFs are prevalent within vertebrate transcriptomes, (ii) the majority show signatures of active translation, and (iii)uORFs act as potent regulators of translation andRNAlevels, with a similar magnitude to miRNAs. Reporter experiments reveal clear repression of downstream translation byuORFs/oORFs.uORFnumber, intercistronic distance, overlap with theCDS, and initiation context most strongly influence translation. Evolution has targeted these features to favoruORFs amenable to regulation over constitutively repressiveuORFs/oORFs. Finally, we observe that the regulatory potential ofuORFs on individual genes is conserved across species. These results provide insight into the regulatory code withinmRNAleader sequences and their capacity to modulate translation across vertebrates. PMID:26896445

  2. Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA

    PubMed Central

    Ozgur, Sezgin

    2014-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 × 10−7M and 1.5 × 10−7M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. IMPORTANCE This work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and

  3. 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

  4. Regulation of Notch-mediated transcription by a bovine herpesvirus 1 encoded protein (ORF2) that is expressed in latently infected sensory neurons.

    PubMed

    Liu, Yilin; Jones, Clinton

    2016-08-01

    Bovine herpesvirus 1 (BoHV-1) is an Alphaherpesvirinae subfamily member that establishes life-long latency in sensory neurons. The latency-related RNA (LR-RNA) is abundantly expressed during latency. An LR mutant virus containing stop codons at the amino-terminus of open reading frame (ORF)2 does not reactivate from latency and replicates less efficiently in tonsils and trigeminal ganglia. ORF2 inhibits apoptosis, interacts with Notch family members, and interferes with Notch-dependent transcription suggesting ORF2 expression enhances survival of infected neurons. The Notch signaling pathway is crucial for neuronal differentiation and survival suggesting that interactions between ORF2 and Notch family members regulate certain aspects of latency. Consequently, for this study, we compared whether ORF2 interfered with the four mammalian Notch family members. ORF2 consistently interfered with Notch1-3-mediated transactivation of three cellular promoters. Conversely, Notch4-mediated transcription was not consistently inhibited by ORF2. Electrophoretic shift mobility assays using four copies of a consensus-DNA binding site for Notch/CSL (core binding factor (CBF)-1, Suppressor of Hairless, Lag-2) as a probe revealed ORF2 interfered with Notch1 and 3 interactions with a CSL family member bound to DNA. Additional studies demonstrated ORF2 enhances neurite sprouting in mouse neuroblastoma cells that express Notch1-3, but not Notch4. Collectively, these studies indicate that ORF2 inhibits Notch-mediated transcription and signaling by interfering with Notch interacting with CSL bound to DNA. PMID:26846632

  5. 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

  6. 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

  7. 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

  8. 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

  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. Transcriptional activator elements for curtovirus C1 expression reside in the 3' coding region of ORF C1.

    PubMed

    Hur, Jingyung; Buckley, Kenneth; Lee, Sukchan; Davis, Keith

    2007-02-28

    Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV), members of curtoviruses, encode seven open reading frames (ORFs) within a approximately 3 kb genome. One of these viral ORFs, C1, is known to play an important role in the early stage of viral infection in plants during initiation of viral DNA replication. We used promoter:: reporter (beta-glucuronidase) gene fusions in transgenic Ara-bidopsis to identify the putative promoter region of BCTV ORF C1. Unlike other geminiviruses, the intergenic region of BCTV was not sufficient to promote C1 expression in transgenic plants. When sequences extending into the coding region of C1 were tested, strong expression of the reporter protein was observed in vascular tissues of transgenic plants. This expression was not dependent on the presence of the intergenic regions or proximal 5' portions of the C1 coding region. Transgenic plants expressing a reporter gene under control of the putative complete C1 promoter were inoculated with virus to determine if any viral transcript affected C1 expression. Virus inoculated plants did not show any altered pattern or change in of reporter gene expression level. These results suggest that (1) important transcriptional activator elements for C1 expression reside in the 3' portion of C1 coding area itself, (2) C1 protein does not auto-regulate its own expression and (3) C1 expression of two curtoviruses is controlled differently compared to other geminiviruses. PMID:17464215

  11. 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

  12. Marek's Disease Viruses Lacking Either R-LORF10 or LORF4 Have Altered Virulence in Chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Marek’s disease virus (MDV) genome encodes about 110 open reading frames (ORFs). Many of these ORFs are annotated based purely on homology to other herpesvirus genes, thus, direct experiments are needed to verify the gene products, especially the hypothetical or MDV-specific ORFs, and character...

  13. 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

  14. The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

    SciTech Connect

    Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.

    2007-09-30

    Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

  15. Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression

    PubMed Central

    Lu, YiQing; Kucharski, Thomas J.; Gamache, Isabelle; Blanchette, Paola; Branton, Philip E.

    2014-01-01

    ABSTRACT Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4−) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4− virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication. IMPORTANCE Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and

  16. The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex.

    PubMed

    Schaecher, Scott R; Diamond, Michael S; Pekosz, Andrew

    2008-10-01

    The severe acute respiratory syndrome coronavirus (SARS-CoV) ORF7b (also called 7b) protein is an integral membrane protein that is translated from a bicistronic open reading frame encoded within subgenomic RNA 7. When expressed independently or during virus infection, ORF7b accumulates in the Golgi compartment, colocalizing with both cis- and trans-Golgi markers. To identify the domains of this protein that are responsible for Golgi localization, we have generated a set of mutant proteins and analyzed their subcellular localizations by indirect immunofluorescence confocal microscopy. The N- and C-terminal sequences are dispensable, but the ORF7b transmembrane domain (TMD) is essential for Golgi compartment localization. When the TMD of human CD4 was replaced with the ORF7b TMD, the resulting chimeric protein localized to the Golgi complex. Scanning alanine mutagenesis identified two regions in the carboxy-terminal portion of the TMD that eliminated the Golgi complex localization of the chimeric CD4 proteins or ORF7b protein. Collectively, these data demonstrate that the Golgi complex retention signal of the ORF7b protein resides solely within the TMD. PMID:18632859

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

    PubMed

    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

  18. 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

  19. 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

  20. The Human Adenovirus E4-ORF1 Protein Subverts Discs Large 1 to Mediate Membrane Recruitment and Dysregulation of Phosphatidylinositol 3-Kinase

    PubMed Central

    Kong, Kathleen; Kumar, Manish; Taruishi, Midori; Javier, Ronald T.

    2014-01-01

    Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K). While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1), a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells. PMID:24788832

  1. GENOME OF HORSEPOX VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 (ITR) and lacked extensive terminal tandem repetition. HSPV contained 236 ORFs with sim...

  2. 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

  3. Varicella-Zoster Virus Open Reading Frame 48 Encodes an Active Nuclease

    PubMed Central

    Mueller, Niklaus H.; Gilden, Don

    2013-01-01

    Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity. PMID:23966396

  4. 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

  5. The complete nucleotide sequence and genomic organization of a novel victorivirus with two non-overlapping ORFs, identified in the plant-pathogenic fungus Phomopsis vexans.

    PubMed

    Zhang, Ru Jia; Zhong, Jie; Shang, Hong Hong; Pan, Xian Ting; Zhu, Hong Jian; Gao, Bi Da

    2015-07-01

    In this study, a novel virus designated Phomopsis vexans RNA virus 1 (PvRV1) was identified in a strain of Phomopsis vexans. The complete genomic nucleotide sequence was determined and analyzed. Sequence analysis indicated that PvRV1 is closely related to viruses in the genus Victorivirus of the family Totiviridae. Two open reading frames (ORF1 and 2) were found in the PvRV1 sequence, and these showed significant similarity to the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), respectively, of members of the family Totiviridae. The two ORFs were spaced 98 nt apart, which is unique to PvRV1 and different from the overlapping arrangement in most victoriviruses. The expression strategies of the CP and RdRp are discussed based on in silico RNA secondary structure analysis. PMID:25902724

  6. Identification and characterization of sORF-encoded polypeptides.

    PubMed

    Chu, Qian; Ma, Jiao; Saghatelian, Alan

    2015-01-01

    Molecular biology, genomics and proteomics methods have been utilized to reveal a non-annotated class of endogenous polypeptides (small proteins and peptides) encoded by short open reading frames (sORFs), or small open reading frames (smORFs). We refer to these polypeptides as s(m)ORF-encoded polypeptides or SEPs. The early SEPs were identified via genetic screens, and many of the RNAs that contain s(m)ORFs were originally considered to be non-coding; however, elegant work in bacteria and flies demonstrated that these s(m)ORFs code for functional polypeptides as small as 11-amino acids in length. The discovery of these initial SEPs led to search for these molecules using methods such as ribosome profiling and proteomics, which have revealed the existence of many SEPs, including novel human SEPs. Unlike screens, omics methods do not necessarily link a SEP to a cellular or biological function, but functional genomic and proteomic strategies have demonstrated that at least some of these newly discovered SEPs have biochemical and cellular functions. Here, we provide an overview of these results and discuss the future directions in this emerging field. PMID:25857697

  7. Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lytic bacteriophages, viruses which infect and lyse bacterial cells, can provide a natural method to reduce bacterial pathogens on produce commodities. The use of multi-phage cocktails is most likely to be effective against bacterial pathogens on produce commodities, and minimize the development of...

  8. Kaposi's Sarcoma-Associated Herpesvirus ORF45 Interacts with Kinesin-2 Transporting Viral Capsid-Tegument Complexes along Microtubules

    PubMed Central

    Sathish, Narayanan; Zhu, Fan Xiu; Yuan, Yan

    2009-01-01

    Open reading frame (ORF) 45 of Kaposi's sarcoma-associated herpesvirus (KSHV) is a tegument protein. A genetic analysis with a null mutant suggested a possible role for this protein in the events leading to viral egress. In this study, ORF45 was found to interact with KIF3A, a kinesin-2 motor protein that transports cargoes along microtubules to cell periphery in a yeast two-hybrid screen. The association was confirmed by both co-immunoprecipitation and immunoflorescence approaches in primary effusion lymphoma cells following virus reactivation. ORF45 principally mediated the docking of entire viral capsid-tegument complexes onto the cargo-binding domain of KIF3A. Microtubules served as the major highways for transportation of these complexes as evidenced by drastically reduced viral titers upon treatment of cells with a microtubule depolymerizer, nocodazole. Confocal microscopic images further revealed close association of viral particles with microtubules. Inhibition of KIF3A–ORF45 interaction either by the use of a headless dominant negative (DN) mutant of KIF3A or through shRNA-mediated silencing of endogenous KIF3A expression noticeably decreased KSHV egress reflecting as appreciable reductions in the release of extracellular virions. Both these approaches, however, failed to impact HSV-1 egress, demonstrating the specificity of KIF3A in KSHV transportation. This study thus reports on transportation of KSHV viral complexes on microtubules by KIF3A, a kinesin motor thus far not implicated in virus transportation. All these findings shed light on the understudied but significant events in the KSHV life cycle, delineating a crucial role of a KSHV tegument protein in cellular transport of viral particles. PMID:19282970

  9. 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.

  10. 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.

  11. Comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses.

    PubMed

    Wiedenheft, Blake; Stedman, Kenneth; Roberto, Francisco; Willits, Deborah; Gleske, Anne-Kathrin; Zoeller, Luisa; Snyder, Jamie; Douglas, Trevor; Young, Mark

    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 spindle-shaped 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 approximately 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. PMID:14747560

  12. 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

  13. The nucleotide sequence and genomic organization of Citrus leaf blotch virus: candidate type species for a new virus genus.

    PubMed

    Vives, M C; Galipienso, L; Navarro, L; Moreno, P; Guerri, J

    2001-08-15

    The complete nucleotide sequence of Citrus leaf blotch virus (CLBV) was determined. CLBV genomic RNA (gRNA) has 8747 nt, excluding the 3'-terminal poly(A) tail, and contains three open reading frames (ORFs) and untranslated regions (UTR) of 73 and 541 nucleotides at the 5' and 3' termini, respectively. ORF1 potentially encodes a 227.4-kDa polypeptide, which has methyltransferase, papain-like protease, helicase, and RNA-dependent RNA polymerase motifs. ORF2 encodes a 40.2-kDa polypeptide containing a motif characteristic of cell-to-cell movement proteins. The 40.7-kDa polypeptide encoded by ORF3 was identified as the coat protein. The genome organization of CLBV resembles that of viruses in the genus Trichovirus, but they differ in various aspects: (i) in trichoviruses ORF2 overlaps ORFs 1 and 3, whereas in CLBV, ORFs 2 and 3 are separated and ORFs 1 and 2 overlap in one nucleotide; (ii) CLBV gRNA and CP are larger than those of trichoviruses; and (iii) the CLBV 3' UTR is larger than that of trichoviruses. Phylogenetic comparisons based on CP amino acid signatures clearly separates CLBV from trichoviruses. Also contrasting with trichoviruses, CLBV could not be transmitted to Chenopodium quinoa Willd. Considering these singularities, we propose that CLBV should be included in a new virus genus. PMID:11504557

  14. 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...

  15. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

    PubMed

    Zhang, Ke; Donnelly, Christopher J; 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; Lloyd, Thomas E; Rothstein, Jeffrey D

    2015-09-01

    The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) 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 orthologue 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 (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, 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 that is amenable to pharmacotherapeutic intervention. PMID:26308891

  16. 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

  17. Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.

    PubMed Central

    Heineman, T C; Cohen, J I

    1994-01-01

    Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir. Images PMID:8151792

  18. 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

  19. 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

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

    PubMed

    Untiveros, Milton; Olspert, Allan; Artola, Katrin; Firth, Andrew E; Kreuze, Jan F; Valkonen, Jari P T

    2016-09-01

    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

  1. 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

  2. Solenopsis invicta virus 3: Mapping of Structural Proteins, Ribosomal Frameshifting, and Similarities to Acyrthosiphon pisum virus and Kelp fly virus

    PubMed Central

    Valles, Steven M.; Bell, Susanne; Firth, Andrew E.

    2014-01-01

    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 structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order. PMID:24686475

  3. Mutational analysis of the monopartite geminivirus beet curly top virus.

    PubMed

    Stanley, J; Latham, J R; Pinner, M S; Bedford, I; Markham, P G

    1992-11-01

    Mutants of the monopartite geminivirus beet curly top virus have been screened for infectivity and symptom development in Nicotiana benthamiana and Beta vulgaris, for replication competence in N. benthamiana leaf discs, and for transmission by the leafhopper Circulifer tenellus. Disruption of open reading frame (ORF) V2 by the introduction of a termination codon resulted in symptomless infection of N. benthamiana associated with low levels of virus and reduced single-stranded (ss) DNA and prevented systemic infection of B. vulgaris. Reduced levels of ssDNA were produced by the mutant in N. benthamiana leaf discs, suggesting that V2 affects the synthesis or accumulation of this viral DNA form. Mutants in which ORF C2 had been truncated by the introduction of termination codons or by frame-shifting remained highly infectious and induced severe symptoms in both N. benthamiana and B. vulgaris. Similarly, a mutant containing a termination codon within ORF C3 was highly infectious and induced severe symptoms in N. benthamiana although infectivity in B. vulgaris was greatly reduced, symptoms were extremely mild, and virus levels were low. A synergistic effect of a double mutation in ORFs C2 and C3, manifested by the inability of mutants to systemically infect N. benthamiana and the production of reduced amounts of ssDNA in N. benthamiana leaf discs, suggests that both ORFs are functional in this host. A mutant containing a termination codon within the 5' terminus of ORF C4 produced severe symptoms in both N. benthamiana and B. vulgaris resembling those induced by wild-type virus. Comparison with the phenotypes of previously characterized ORF C4 mutants suggests that a conserved core sequence of this ORF is an important symptom determinant. ORF C2, C3, and C4 mutants produced virus particles and were transmitted by C. tenellus, eliminating agroinoculation as a contributory factor to the mutant phenotypes. Our results are compared with those derived from mutagenesis studies

  4. 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

  5. Examination of potato virus X proteins synthesized in infected tobacco plants.

    PubMed

    Price, M

    1992-09-01

    Nucleotide sequence analysis of potato virus X (PVX) genomic RNA predicts five open reading frames (ORFs). Previous analysis of total RNAs from PVX-infected leaf tissue suggested that six subgenomic RNAs are synthesized during infection. However, the proteins encoded by the genomic RNA, the subgenomic RNAs, or the predicted ORFs have not been identified in vivo. To characterize the coding properties of the viral RNA, particularly to determine whether the five predicted ORFs function in vivo, total protein extracts prepared from PVX-infected leaf tissue were analyzed by using antibodies raised against virus-specific synthetic peptides and against the virus capsid protein. Dot blot analyses showed that these antibodies reacted to PVX-infected extracts, indicating in vivo expression of the five predicted ORFs. In addition, Western blot (immunoblot) analysis of the extracts showed that ORF 1, 2, 3, and 4 peptide antisera and coat protein antiserum detect predominantly a single protein. PMID:1501297

  6. Analysis of synonymous codon usage in porcine reproductive and respiratory syndrome virus.

    PubMed

    Liu, Yong-sheng; Zhou, Jian-hua; Chen, Hao-tai; Ma, Li-na; Ding, Yao-zhong; Wang, Meng; Zhang, Jie

    2010-08-01

    In this study, we calculated the relative synonymous codon usage (RSCU) values and codon usage bias (CUB) values to implement a comparative analysis of codon usage pattern of open reading frames (ORFs) which belong to the two main genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). By analysis of synonymous codon usage values in each ORF of PRRSV, the optimal codons for most amino acids were all C or G-ended codons except GAU for Asp, CAU for His, UUU for Phe and CCU for Pro. The synonymous codon usage patterns in different ORFs of PRRSV were different and genetically conserved. Among them, ORF1a, ORF4, ORF5 and ORF7 could cluster these strains into the two main serotypes (EU and US). Due to mutational pressure, compositional constraint played an important role in shaping the synonymous codon usage pattern in different ORFs, and the synonymous codon usage diversity in ORFs was correlated with gene function. The degree of CUB for some particular amino acids under strong selection pressure probably served as a potential genetic marker for each ORF in PRRSV. However, gene length and translational selection in nature had no effect on the synonymous codon usage pattern in PRRSV. These conclusions could not only offer an insight into the synonymous codon usage pattern and differentiation of gene function, but also assist in understanding the discrepancy of evolution among ORFs in PRRSV. PMID:20438864

  7. Complete genome sequence of yacon necrotic mottle virus, a novel putative member of the genus Badnavirus.

    PubMed

    Lee, Ye-Ji; Kwak, Hae-Ryun; Lee, Young-Kee; Kim, Mi-Kyeong; Choi, Hong-Soo; Seo, Jang-Kyun

    2015-04-01

    The complete genome sequence of a previously undescribed virus isolated from a yacon plant exhibiting necrotic mottle, chlorosis, stunting, and leaf malformation symptoms in Gyeongju, Korea, was determined. The genome of this virus consists of one circular double-stranded DNA of 7661 bp in size. The genome contained four open reading frames (ORFs 1 to 4) on the plus strand that potentially encode proteins of 26, 32, 234, and 25 kDa. Protein BLAST analysis showed that ORF3, which is the largest ORF, has 45 % amino acid sequence identity (with 89 % coverage) to the ORF3 of fig badnavirus 1 (FBV-1), a recently identified badnavirus. Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Badnavirus. The name yacon necrotic mottle virus (YNMoV) is proposed for this new virus. PMID:25643816

  8. Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

    PubMed

    Wirth, Jennifer Fulton; Snyder, Jamie C; Hochstein, Rebecca A; Ortmann, Alice C; Willits, Deborah A; Douglas, Trevor; Young, Mark J

    2011-06-20

    Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication. PMID:21496857

  9. Comparative Full Length Sequence Analysis of Oncogenic and Vaccine (Rispens) Strains of Marek's Disease Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete DNA sequence of the Marek’s disease virus serotype 1 vaccine strain CVI988 was determined and consists of 178,311 bp with an overall gene organization identical to that of the oncogenic strains. In examining open reading frames (ORFs), nine ORFs differ between vaccine and oncogenic stra...

  10. 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

  11. Reverse genetic manipulation of the overlapping coding regions for structural proteins of the type II porcine reproductive and respiratory syndrome virus.

    PubMed

    Yu, Dandan; Lv, Jian; Sun, Zhi; Zheng, Haihong; Lu, Jiaqi; Yuan, Shishan

    2009-01-01

    The overlapping genomic regions coding for structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) poses problems for molecular dissection of the virus replication process. We constructed five mutant full-length cDNA clones with the overlapping regions unwound and 1 to 3 restriction sites inserted between two adjacent ORFs (ORF1/2, ORF4/5, ORF5/6, ORF 6/7 and ORF7/3' UTR), which generated the recombinant viruses. Our findings demonstrated that 1) the overlapping structural protein ORFs can be physically separated, and is dispensable for virus viability; 2) such ORF separations did not interrupt the subgenomic RNA synthesis; 3) the plaque morphology, growth kinetics, and antigenicity of these mutant viruses were virtually indistinguishable from those of the parental virus in cultured cells; and 4) these mutant viruses remained genetic stable in vitro. This study lays a foundation for further molecular dissection of PRRSV replication process, and development of genetically tagged vaccines against PRRS. PMID:18977502

  12. Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72.

    PubMed

    van Blitterswijk, Marka; Gendron, Tania F; Baker, Matthew C; DeJesus-Hernandez, Mariely; Finch, NiCole A; Brown, Patricia H; Daughrity, Lillian M; Murray, Melissa E; Heckman, Michael G; Jiang, Jie; Lagier-Tourenne, Clotilde; Edbauer, Dieter; Cleveland, Don W; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Petrucelli, Leonard; Boeve, Bradley F; Graff-Radford, Neill R; Boylan, Kevin B; Dickson, Dennis W; Rademakers, Rosa

    2015-12-01

    The loss of chromosome 9 open reading frame 72 (C9ORF72) expression, associated with C9ORF72 repeat expansions, has not been examined systematically. Three C9ORF72 transcript variants have been described thus far; the GGGGCC repeat is located between two non-coding exons (exon 1a and exon 1b) in the promoter region of transcript variant 2 (NM_018325.4) or in the first intron of variant 1 (NM_145005.6) and variant 3 (NM_001256054.2). We studied C9ORF72 expression in expansion carriers (n = 56) for whom cerebellum and/or frontal cortex was available. Using quantitative real-time PCR and digital molecular barcoding techniques, we assessed total C9ORF72 transcripts, variant 1, variant 2, variant 3, and intron containing transcripts [upstream of the expansion (intron 1a) and downstream of the expansion (intron 1b)]; the latter were correlated with levels of poly(GP) and poly(GA) proteins aberrantly translated from the expansion as measured by immunoassay (n = 50). We detected a decrease in expansion carriers as compared to controls for total C9ORF72 transcripts, variant 1, and variant 2: the strongest association was observed for variant 2 (quantitative real-time PCR cerebellum: median 43 %, p = 1.26e-06, and frontal cortex: median 58 %, p = 1.11e-05; digital molecular barcoding cerebellum: median 31 %, p = 5.23e-10, and frontal cortex: median 53 %, p = 5.07e-10). Importantly, we revealed that variant 1 levels greater than the 25th percentile conferred a survival advantage [digital molecular barcoding cerebellum: hazard ratio (HR) 0.31, p = 0.003, and frontal cortex: HR 0.23, p = 0.0001]. When focusing on intron containing transcripts, analysis of the frontal cortex revealed an increase of potentially truncated transcripts in expansion carriers as compared to controls [digital molecular barcoding frontal cortex (intron 1a): median 272 %, p = 0.003], with the highest levels in patients pathologically diagnosed with frontotemporal lobar degeneration

  13. 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

  14. Expression of the C-terminal ORF2 protein of duck astrovirus for application in a serological test.

    PubMed

    Wang, Xiaoyan; Wang, Yuzhou; Xie, Xiaoyu; Zhang, Bing; Zhang, Dabing

    2011-01-01

    Duck astrovirus (DAstV) is an important pathogen causing duck viral hepatitis (DVH), a highly contagious and fatal disease in young ducklings. To provide an antigen for a diagnostic serum test, the C-terminus of DAstV ORF2 protein was expressed in Escherichia coli. Four positive and 30 negative sera were used to validate the purified ORF2 protein by developing an indirect enzyme-linked immunosorbent assay (ELISA). No cross-reactions were found against other duck pathogens, including duck hepatitis A virus, duck plague herpesvirus, duck reovirus, Newcastle disease virus, and Riemerella anatipestifer 12/19 (63.2%) and 26/51 (51%) sera samples from two flocks of ducks that survived DAstV infections in commercial duck farms were positive for DAstV by this method, respectively. Interestingly, DAstV-specific antibodies were also detected in 12 (28.6%) of 42 sera samples from a different flock without DVH, indicating a wide distribution of subclinical infections caused by DAstV. PMID:20863856

  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. PMID:2602148

  16. 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

  17. 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

  18. ORF3 of duck circovirus: a novel protein with apoptotic activity.

    PubMed

    Xiang, Qi-Wang; Wang, Xin; Xie, Zhi-Jing; Sun, Ya-Ni; Zhu, Yan-Li; Wang, Shu-Jing; Liu, Hong-Jie; Jiang, Shi-Jin

    2012-09-14

    Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (P<0.05) that of the Sf9 cells infected with wild-type baculovirus at 24, 48 and 72 h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity. PMID:22537707

  19. Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein

    PubMed Central

    Xu, Han; Wang, Tao; Zhang, Yanming

    2015-01-01

    Porcine circovirus type 2 (PCV2) is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs). Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER), is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn. PMID:26035722

  20. 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

  1. 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

  2. Production of Myxoma Virus Gateway Entry and Expression Libraries and Validation of Viral Protein Expression

    PubMed Central

    Smallwood, Sherin E.; Rahman, Masmudur M.; Werden, Steven J.; Martino, Maria Fernanda; McFadden, Grant

    2011-01-01

    Invitrogen’s Gateway technology is a recombination-based cloning method that allows for rapid transfer of numerous open reading frames (ORFs) into multiple plasmid vectors, making it useful for diverse high-throughput applications. Gateway technology has been utilized to create an ORF library for Myxoma virus (MYXV), a member of the Poxviridae family of DNA viruses. MYXV is the prototype virus for the genus Leporipoxvirus, and is pathogenic only in European rabbits. MYXV replicates exclusively in the host cell cytoplasm, and its genome encodes 171 ORFs. A number of these ORFs encode proteins that interfere with or modulate host defense mechanisms, particularly the inflammatory responses. Furthermore, MYXV is able to productively infect a variety of human cancer cell lines and is being developed as an oncolytic virus for treating human cancers. MYXV is therefore an excellent model for studying poxvirus biology, pathogenesis, and host tropism, and a good candidate for ORFeome development. PMID:21538302

  3. Sequence comparison of JSRV with endogenous proviruses: envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor.

    PubMed

    Bai, J; Bishop, J V; Carlson, J O; DeMartini, J C

    1999-06-01

    Ovine pulmonary carcinoma, a contagious lung cancer of sheep, is caused by the oncogenic jaagsiekte sheep retrovirus (JSRV) that is closely related to a family of endogenous sheep retroviral sequences (ESRVs). By using exogenous virus-specific U3 oligonucleotide primers, the entire JSRV proviral genome or its 3' part was amplified from tumor DNA. Analysis of these proviral sequences revealed a novel open reading frame (ORF) within the pol coding region, designated ORF X, which was well conserved in ESRV and JSRV sequences. Deduced amino acids of ORF X showed similarity to a portion of the mammalian adenosine receptor subtype 3, a member of the G-protein-coupled receptor family. Comparison of deduced env amino acids of six JSRV strains from three continents identified 15 residues that defined two distinct genotypes of JSRVs. Sequence analysis identified two highly variable regions between JSRV and ESRV in the transmembrane domain of env (TM) and the 3' unique sequence (U3) of the long terminal repeat, from which JSRV-specific DNA probes were derived. By using these DNA probes in Southern hybridization, for the first time we successfully identified JSRV proviral sequences in tumor genomic DNA in the presence of multiple ESRV loci, validating the use of exogenous virus-specific DNA probes in the analysis of oncogenic proviral integration sites and identification of integrated exogenous proviral sequences. PMID:10366570

  4. 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

  5. Full coding hepatitis E virus genotype 3 genome amplification method.

    PubMed

    Muñoz-Chimeno, M; Forero, J E; Echevarría, J M; Muñoz-Bellido, J L; Vázquez-López, L; Morago, L; García-Galera, M C; Avellón, A

    2016-04-01

    Hepatitis E virus (HEV) genotype 3 produces zoonotic infection associated with the consumption of infected animals. HEV infections can become chronic in immunocompromised (IC) patients. The viral genome has three well defined open reading frames (ORF1, ORF2 and ORF3) within which various domains and functions have been described. This paper (i) describes a new method of complete sequencing of the HEV coding region through overlapping PCR systems, (ii) establishes a consensus sequence and polymorphic positions (PP) for each domain, and (iii) analyzes the complete coding sequence of an IC patient. With regard to the consensus, a high percentage of PP was observed in protease (PP=19%) and the X domain (PP=22%) within ORF1, the N-terminal region of the S domain (PP=22%) in ORF2, and the P1 (PP=35%) and P2 (PP=25%) domains in ORF3. In contrast, the ORF1 Y, ORF2 S, ORF2 M and ORF3 D1 domains were conserved in the reference sequences (0.40, 1, 0.70 and 0% of PP, respectively). The sequence from the IC patient had more mutations in the RpRp (D1235G, Q1242R, S1454T, V1480I, I1502 V, K1511R, G1373 V, E1442D, V1693 M), the terminal ORF2 S- domain (F10L, S26T, G36S, S70P, A105 V, I113 V), the X domain (T938 M, T856 V, S898A) and the helicase (S1014N, S975T, Q1133 K). PMID:26784284

  6. 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.

  7. 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

  8. Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

    PubMed

    Qi, Ying; Zhang, Feng; Zhang, Li; Harrison, Tim J; Huang, Weijin; Zhao, Chenyan; Kong, Wei; Jiang, Chunlai; Wang, Youchun

    2015-01-01

    The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope. PMID:26161670

  9. 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.

  10. A Biomedically Enriched Collection of 7000 Human ORF Clones

    PubMed Central

    Rolfs, Andreas; Hu, Yanhui; Ebert, Lars; Hoffmann, Dietmar; Zuo, Dongmei; Ramachandran, Niro; Raphael, Jacob; Kelley, Fontina; McCarron, Seamus; Jepson, Daniel A.; Shen, Binghua; Baqui, Munira M. A.; Pearlberg, Joseph; Taycher, Elena; DeLoughery, Craig; Hoerlein, Andreas; Korn, Bernhard; LaBaer, Joshua

    2008-01-01

    We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in ∼15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance. PMID:18231609

  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. Human herpesvirus 8 infections in the Amsterdam Cohort Studies (1984–1997): Analysis of seroconversions to ORF65 and ORF73

    PubMed Central

    Goudsmit, Jaap; Renwick, Neil; Dukers, Nicole H. T. M.; Coutinho, Roel A.; Heisterkamp, Siem; Bakker, Margreet; Schulz, Thomas F.; Cornelissen, Marion; Weverling, Gerrit J.

    2000-01-01

    We have shown previously that human herpesvirus 8 (HHV8) seroconversion for antibodies to the latency-associated nuclear antigen encoded by ORF73 and/or the lytic capsid antigen (vp19) encoded by ORF65 is associated with orogenital contact and is strongly linked to the development of Kaposi's sarcoma among HIV-infected individuals in the Amsterdam Cohort Studies. Here, we investigate the relationship between seroconversion to these antigens and primary HHV8 infection. Between 1984 and 1997, 215 HHV8 seroconversions to ORF73 (106 cases or 49%) and/or to ORF65 (159 cases or 74%) were recorded in the cohort of homosexual men. The HHV8 seroconversion rate among HIV-infected homosexual men (6.2 per 100 person years) was consistently higher than among HIV-uninfected men (2.6 per 100 person years). In HIV-infected but not in uninfected individuals, seroconversion to ORF73/latency-associated nuclear antigen precedes that to ORF65/vp19. Antibody levels to both ORF65- and ORF73-encoded antigens were higher in HIV-infected than in HIV-uninfected men, and among HIV-seropositives, antibody levels to ORF65/vp19 rise even higher with declining CD4 cell counts and peak with Kaposi's sarcoma development, suggesting continuing and increasing viral replication. In 10.3% of HHV8 seroconversions, transient serum viremia could be demonstrated before or at seroconversion. Together with the previously reported link between unprotected orogenital sex and HHV8 seroconversion, our observations suggest that HHV8 seroconversions result from primary infections. PMID:10781089

  13. Detection and molecular characterization of a Grapevine Roditis leaf discoloration-associated virus (GRLDaV) variant in an autochthonous grape from Apulia (Italy).

    PubMed

    Chiumenti, Michela; Giampetruzzi, Annalisa; Morelli, Massimiliano; Savino, Vito Nicola; Martelli, Giovanni Paolo; La Notte, Pierfederico; Palmisano, Francesco; Saldarelli, Pasquale

    2016-06-01

    The complete nucleotide sequence and genome organization of a new Badnavirus isolated from the autochthonous grapevine variety "Bombino nero" from Apulia (Italy) was determined. The genome of this virus consists of 7097 nt and has four open reading frames (ORFs). Analysis of putative proteins encoded by each ORF revealed greatest sequence similarity to Grapevine Roditis leaf discoloration-associated virus w4 (GRLDaV; NC_027131). In a pairwise alignment with GLRDaV w4 genome sequence, the "Bombino Nero" sequence was 109 nt longer with a major 57 nt insertion between positions 2405 and 2413. Furthermore, its putative ORF4 is located after the ORF3, while in the GLRDaV w4 sequence, the putative ORF4 completely overlapped ORF3. Nucleotide analysis classifies this new Badnavirus as a GLRDaV strain, which was named GRLDaV-BN. Multi-year field observations showed that the GLRDaV-BN-infected vine was symptomless. PMID:26924587

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 and conceptual translations of virus species in the genus Carmovirus, family Tombusviridae. The orde...

  15. The expanding biology of the C9orf72 nucleotide repeat expansion in neurodegenerative disease.

    PubMed

    Haeusler, Aaron R; Donnelly, Christopher J; Rothstein, Jeffrey D

    2016-06-01

    A nucleotide repeat expansion (NRE) within the chromosome 9 open reading frame 72 (C9orf72) gene was the first of this type of mutation to be linked to multiple neurological conditions, including amyotrophic lateral sclerosis and frontotemporal dementia. The pathogenic mechanisms through which the C9orf72 NRE contributes to these disorders include loss of C9orf72 function and gain-of-function mechanisms of C9orf72 driven by toxic RNA and protein species encoded by the NRE. These mechanisms have been linked to several cellular defects - including nucleocytoplasmic trafficking deficits and nuclear stress - that have been observed in both patients and animal models. PMID:27150398

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

    PubMed

    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

    2014-07-01

    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

  17. 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

  18. 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

  19. 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

  20. Generation of a Genome Scale Lentiviral Vector Library for EF1α Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer

    PubMed Central

    Škalamera, Dubravka; Dahmer, Mareike; Purdon, Amy S.; Wilson, Benjamin M.; Ranall, Max V.; Blumenthal, Antje; Gabrielli, Brian; Gonda, Thomas J.

    2012-01-01

    The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors. PMID:23251614

  1. The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

    PubMed

    Music, Nedzad; Gagnon, Carl A

    2010-12-01

    Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis. PMID:20388230

  2. Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

    PubMed Central

    Nevels, Michael; Täuber, Birgitt; Kremmer, Elisabeth; Spruss, Thilo; Wolf, Hans; Dobner, Thomas

    1999-01-01

    Previous observations that the adenovirus type 5 (Ad5) E4orf6 and E4orf3 gene products have redundant effects in viral lytic infection together with the recent findings that E4orf6 possesses transforming potential prompted us to investigate the effect of E4orf3 expression on the transformation of primary rat cells in combination with adenovirus E1 oncogene products. Our results demonstrate for the first time that E4orf3 can cooperate with adenovirus E1A and E1A plus E1B proteins to transform primary baby rat kidney cells, acting synergistically with E4orf6 in the presence of E1B gene products. Transformed rat cells expressing E4orf3 exhibit morphological alterations, higher growth rates and saturation densities, and increased tumorigenicity compared with transformants expressing E1 proteins only. Consistent with previous results for adenovirus-infected cells, the E4orf3 protein is immunologically restricted to discrete nuclear structures known as PML oncogenic domains (PODs) in transformed rat cells. As opposed to E4orf6, the ability of E4orf3 to promote oncogenic cell growth is probably not linked to a modulation of p53 functions and stability. Instead, our results indicate that the transforming activities of E4orf3 are due to combinatorial effects that involve the binding to the adenovirus 55-kDa E1B protein and the colocalization with PODs independent from interactions with the PML gene product. These data fit well with a model in which the reorganization of PODs may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. In sum, our results provide strong evidence for the idea that interactions with PODs by viral proteins are linked to oncogenic transformation. PMID:9882365

  3. A novel virus transmitted through pollination causes ring-spot disease on gentian (Gentiana triflora) ovaries.

    PubMed

    Atsumi, Go; Tomita, Reiko; Yamashita, Tetsuro; Sekine, Ken-Taro

    2015-02-01

    In this study, we identified a novel virus from gentian (Gentiana triflora) that causes ring-spots on ovaries. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. Pollen grains carrying the virus were used to infect host plants by hand-pollination. RNA extracted from purified virions indicated that the virus had two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1-3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence similarity and phylogenetic analysis suggested that the virus is closely related to members of the genera Pecluvirus and Hordeivirus but distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus gentian ovary ring-spot virus. PMID:25351517

  4. Expression Profiling of WSSV ORF 199 and Shrimp Ubiquitin Conjugating Enzyme in WSSV Infected Penaeus monodon

    PubMed Central

    Jeena, K.; Prasad, K. Pani; Pathan, Mujahid Khan; Babu, P. Gireesh

    2012-01-01

    White spot syndrome virus (WSSV) is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF) 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc) was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1α was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i) was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry. PMID:25049679

  5. Nyamanini and Midway Viruses Define a Novel Taxon of RNA Viruses in the Order Mononegavirales▿ †

    PubMed Central

    Mihindukulasuriya, Kathie A.; Nguyen, Nang L.; Wu, Guang; Huang, Henry V.; Travassos da Rosa, Amelia P. A.; Popov, Vsevolod L.; Tesh, Robert B.; Wang, David

    2009-01-01

    Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order. PMID:19279111

  6. 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...

  7. 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

  8. Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion☆

    PubMed Central

    Waite, Adrian J.; Bäumer, Dirk; East, Simon; Neal, James; Morris, Huw R.; Ansorge, Olaf; Blake, Derek J.

    2014-01-01

    An intronic G4C2 hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250–1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype–phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease. PMID:24559645

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

    PubMed

    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

  10. 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…

  11. 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

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

    PubMed

    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

  13. Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR)

    PubMed Central

    Christian, Claiborne M.; deHaro, Dawn; Kines, Kristine J.; Sokolowski, Mark; Belancio, Victoria P.

    2016-01-01

    Long Interspersed Element 1 (LINE-1 or L1) is capable of causing genomic instability through the activity of the L1 ORF2 protein (ORF2p). This protein contains endonuclease (EN) and reverse transcriptase (RT) domains that are necessary for the retrotransposition of L1 and the Short Interspersed Element (SINE) Alu. The functional importance of approximately 50% of the ORF2p molecule remains unknown, but some of these sequences could play a role in retrotransposition, or be necessary for the enzymatic activities of the EN and/or RT domains. Conventional approaches using the full-length, contiguous ORF2p make it difficult to study the involvement of these unannotated sequences in the function of L1 ORF2p. Our lab has developed a Bipartile Alu Retrotransposition (BAR) assay that relies on separate truncated ORF2p fragments: an EN-containing and an RT-containing fragment. We validated the utility of this method for studying the ORF2p function in retrotransposition by assessing the effect of expression levels and previously characterized mutations on BAR. Using BAR, we identified two pairs of amino acids important for retrotransposition, an FF and a WD. The WD appears to play a role in cDNA synthesis by the ORF2p molecule, despite being outside the canonical RT domain. PMID:27095191

  14. 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

  15. Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR).

    PubMed

    Christian, Claiborne M; deHaro, Dawn; Kines, Kristine J; Sokolowski, Mark; Belancio, Victoria P

    2016-06-01

    Long Interspersed Element 1 (LINE-1 or L1) is capable of causing genomic instability through the activity of the L1 ORF2 protein (ORF2p). This protein contains endonuclease (EN) and reverse transcriptase (RT) domains that are necessary for the retrotransposition of L1 and the Short Interspersed Element (SINE) Alu. The functional importance of approximately 50% of the ORF2p molecule remains unknown, but some of these sequences could play a role in retrotransposition, or be necessary for the enzymatic activities of the EN and/or RT domains. Conventional approaches using the full-length, contiguous ORF2p make it difficult to study the involvement of these unannotated sequences in the function of L1 ORF2p. Our lab has developed a Bipartile Alu Retrotransposition (BAR) assay that relies on separate truncated ORF2p fragments: an EN-containing and an RT-containing fragment. We validated the utility of this method for studying the ORF2p function in retrotransposition by assessing the effect of expression levels and previously characterized mutations on BAR. Using BAR, we identified two pairs of amino acids important for retrotransposition, an FF and a WD. The WD appears to play a role in cDNA synthesis by the ORF2p molecule, despite being outside the canonical RT domain. PMID:27095191

  16. 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

  17. 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.

  18. Transcriptional analysis of the virion-sense genes of the geminivirus beet curly top virus.

    PubMed

    Frischmuth, S; Frischmuth, T; Latham, J R; Stanley, J

    1993-11-01

    The genome of the geminivirus beet curly top virus (BCTV) consists of a single circular DNA containing overlapping open reading frames (ORFs) located on both the virion-sense and complementary-sense DNA strands. To investigate the expression of these ORFs, RNA extracted from infected Nicotiana benthamiana and Beta vulgaris has been examined for the presence of viral transcripts. An abundant 1.1-kb virion-sense polyadenylated RNA and four complementary-sense polyadenylated RNAs of 1.7, 1.5, 1.3, and 0.7 kb have been identified by northern blot hybridization, confirming the bidirectional transcription strategy implied by the arrangement of ORFs. We previously demonstrated that two overlapping virion-sense ORFs are involved in coat protein synthesis (ORF V1) and viral single-stranded DNA accumulation (ORF V2). Mutants of a third virion-sense ORF (ORF V3), located upstream and overlapping ORFs V1 and V2, retain the ability to replicate efficiently in N. benthamiana leaf discs but produce an asymptomatic infection in N. benthamiana and B. vulgaris at low frequency, associated with reduced levels of viral DNA compared to wild-type infection. Our data support the recent suggestion that ORF V3 participates in virus movement. The 1.1 kb virion-sense RNA comprises a population of overlapping transcripts with 5' termini suitably positioned for the expression of ORFs V1, V2, and V3. The overlapping arrangement of the transcripts and juxtaposition of putative regulatory elements could provide a means for the temporal control of virion-sense gene expression. PMID:7692668

  19. 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

  20. 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.

  1. RUNX1 Permits E4orf6-Directed Nuclear Localization of the Adenovirus E1B-55K Protein and Associates with Centers of Viral DNA and RNA Synthesis▿

    PubMed Central

    Marshall, Leslie J.; Moore, Amy C.; Ohki, Misao; Kitabayashi, Issay; Patterson, David; Ornelles, David A.

    2008-01-01

    The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-ΔN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1. PMID:18417565

  2. The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family.

    PubMed

    Johnson, K N; Christian, P D

    1998-01-01

    The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family. PMID:9460942

  3. Modified Indirect Porcine Circovirus (PCV) Type 2-Based and Recombinant Capsid Protein (ORF2)-Based Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to PCV

    PubMed Central

    Nawagitgul, Porntippa; Harms, Perry A.; Morozov, Igor; Thacker, Brad J.; Sorden, Steven D.; Lekcharoensuk, Chalermpol; Paul, Prem S.

    2002-01-01

    Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations. PMID:11777826

  4. Xiburema Virus, a Hitherto Undescribed Virus within the Family Rhabdoviridae Isolated in the Brazilian Amazon Region

    PubMed Central

    Martins, Lívia C.; Diniz Júnior, José Antonio P.; de Almeida Medeiros, Daniele Barbosa; Cardoso, Jedson F.; da Silva, Daisy E. A.; de Oliveira, Layanna F.; de Vasconcelos, Janaina M.; Vianez Júnior, João Lídio da S. G.; Vasconcelos, Pedro F. C.

    2014-01-01

    We report here the first complete open reading frame (ORF) genome sequence of Xiburema virus (XIBV), that of strain BE AR362159, isolated from mosquitoes (Sabethes intermedius) in Sena Madureira, Acre state, northern Brazil. All genes showed similarities with those belonging to members of the family Rhabdoviridae. PMID:24948755

  5. A hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic ALS in Taiwan.

    PubMed

    Tsai, Ching-Paio; Soong, Bing-Wen; Tu, Pang-Hsien; Lin, Kon-Ping; Fuh, Jong-Ling; Tsai, Pei-Chien; Lu, Yi-Chun; Lee, I-Hui; Lee, Yi-Chung

    2012-09-01

    A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene was recently identified as an important cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasian populations. The role of the C9ORF72 repeat expansion in ALS in Chinese populations has received little attention. We therefore performed mutation analyses in a Taiwanese cohort of 22 unrelated familial ALS (FALS) patients and 102 sporadic ALS patients of Han Chinese descent. The C9ORF72 mutation was found in 4 FALS (18.2%; 4/22) and 2 sporadic ALS patients (2.0%; 2/102). The C9ORF72 repeat numbers in the 300 healthy controls and the 118 ALS patients without the C9ORF72 mutation ranged from 3 to 15. Needle biopsy in the left frontal cortex of 1 patient with FALS-frontotemporal dementia revealed numerous cytoplasmic TAR DNA-binding protein 43 (TDP-43) inclusions and minimal neuritis, consistent with type B frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) pathology. This study clearly demonstrates the existence and importance of the C9ORF72 hexanucleotide repeat expansion in a Taiwanese ALS cohort of Chinese origin, and supports the global presence of the C9ORF72 repeat expansion in ALS. PMID:22673113

  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. FASTA barcodes: a simple method for the identification of yeast ORF deletions.

    PubMed

    McMahon, K Wyatt; Manukyan, Arkadi; Dungrawala, Huzefa; Montgomery, Micah; Nordstrom, Brian; Wright, Jill; Abraham, Lesley; Schneider, Brandt L

    2011-09-01

    A consortium of yeast geneticists have created -6000 individual ORF deletions, representing > 96% of the currently verified or predicted ORFs in S. cerevisiae. Importantly, molecular barcodes (each a unique 20 bp sequence termed either Uptag or Downtag) were used as identifiers for every ORF deletion. Microarray analyses of pooled yeast deletions has been used to identify thousands of genes involved in general fitness, haploinsufficiency, drug resistance and DNA damage repair. However, application of this powerful technology requires considerable expense, expertise and specialized equipment. While standard PCR techniques and specifically designed PCR primers can be used to confirm that a given ORF is in fact deleted, this procedure cannot be used to identify unknown deletions. In theory, every ORF deletion could be determined by barcode sequencing. However, neither a consolidated barcode database nor a reliable search engine is currently available for this purpose. To address this need, we have adapted a FASTA sequence program that utilizes the unique barcode database to allow users to identify individual ORF deletions, based upon simple sequencing reactions of PCR amplifications of either Uptag or Downtag barcodes. In silico and practical testing of this application reveals that it is an inexpensive, reliable and reproducible method for rapidly identifying unknown deletions. This approach allows laboratories to conduct small- or large-scale genetic screens with pooled yeast deletion strains and identify or verify any ORF deletion without the need for microarray technology. PMID:21809386

  9. 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

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

    PubMed

    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

    2015-09-15

    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

  11. Tau pathology in frontotemporal lobar degeneration with C9ORF72 hexanucleotide repeat expansion.

    PubMed

    Bieniek, Kevin F; Murray, Melissa E; Rutherford, Nicola J; Castanedes-Casey, Monica; DeJesus-Hernandez, Mariely; Liesinger, Amanda M; Baker, Matthew C; Boylan, Kevin B; Rademakers, Rosa; Dickson, Dennis W

    2013-02-01

    An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration associated with TDP-43 pathology (FTLD-TDP). In addition to TDP-43-positive neuronal and glial inclusions, C9ORF72-linked FTLD-TDP has characteristic TDP-43-negative neuronal cytoplasmic and intranuclear inclusions as well as dystrophic neurites in the hippocampus and cerebellum. These lesions are immunopositive for ubiquitin and ubiquitin-binding proteins, such as sequestosome-1/p62 and ubiquilin-2. Studies examining the frequency of the C9ORF72 mutation in clinically probable Alzheimer's disease (AD) have found a small proportion of AD cases with the mutation. This prompted us to systematically explore the frequency of Alzheimer-type pathology in a series of 17 FTLD-TDP cases with mutations in C9ORF72 (FTLD-C9ORF72). We identified four cases with sufficient Alzheimer-type pathology to meet criteria for intermediate-to-high-likelihood AD. We compared AD pathology in the 17 FTLD-C9ORF72 to 13 cases of FTLD-TDP linked to mutations in the gene for progranulin (FTLD-GRN) and 36 cases of sporadic FTLD (sFTLD). FTLD-C9ORF72 cases had higher Braak neurofibrillary tangle stage than FTLD-GRN. Increased tau pathology in FTLD-C9ORF72 was assessed with thioflavin-S fluorescent microscopy-based neurofibrillary tangle counts and with image analysis of tau burden in temporal cortex and hippocampus. FTLD-C9ORF72 had significantly more neurofibrillary tangles and higher tau burden compared with FTLD-GRN. The differences were most marked in limbic regions. On the other hand, sFTLD and FTLD-C9ORF72 had a similar burden of tau pathology. These results suggest FTLD-C9ORF72 has increased propensity for tau pathology compared to FTLD-GRN, but not sFTLD. The accumulation of tau as well as lesions immunoreactive for ubiquitin and ubiquitin-binding proteins (p62 and ubiquilin-2) suggests that mutations in C9ORF72 may involve disrupted

  12. 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

  13. Complete nucleotide sequence of the new potexvirus "Alstroemeria virus X". Brief report.

    PubMed

    Fuji, S; Shinoda, K; Ikeda, M; Furuya, H; Naito, H; Fukumoto, F

    2005-11-01

    A flexuous virus was isolated in Japan from an alstroemeria plant showing mosaic symptoms. The virus had a broad host range but had systemically latent infectivity in alstroemeria. The virus was assigned to the genus Potexvirus based on morphology and physical properties and on an analysis of the complete nucleotide sequence. The genomic RNA of the virus was 7,009 nucleotides in length, excluding the 3'-terminal poly (A) tail. It contained five open reading frames (ORFs), which was consistent with other members of the genus Potexvirus. Although nucleotide sequences of the ORFs differ from previously reported potexviruses, a phylogenetic analysis placed it phylogenetically close to Narcissus mosaic virus and Scallion virus X. Therefore, we propose that this virus should be designated as Alstroemeria virus X (AlsVX). PMID:15986173

  14. Phenotypic properties of herpes simplex virus 1 containing a derepressed open reading frame P gene.

    PubMed Central

    Lagunoff, M; Randall, G; Roizman, B

    1996-01-01

    Open reading frame P (ORF P) maps in the viral DNA sequences transcribed during latency and is located antisense to the gamma 1 34.5 gene. Earlier studies have shown that the expression of ORF P is repressed by an infected cell protein no. 4 binding site straddling the transcription initiation site. We have made monospecific polyclonal antibodies to the protein and constructed a virus, designated ORF P++, in which the infected cell protein no. 4 binding site has been mutagenized, thereby allowing full expression of an unmodified ORF P gene from its natural promoter. We report the following findings. (i) The native protein forms multiple bands on denaturing polyacrylamide gels suggestive of extensive processing and aggregation of the protein; (ii) the protein accumulates in the nucleus in rod-shaped structures perpendicular to the axis of attachment of the infected cell to the solid matrix; (iii) the virus was highly attenuated on inoculation into mice by the intracerebral or ocular route, and virus was not recovered upon explantation of trigeminal ganglia; (iv) although protein synthesis was not prematurely shut off in the human neuroblastoma cell line SK-N-SH, gamma 1 34.5 protein was not detected in immunoblasts. Analyses of electrophoretically separated denatured RNAs indicated that in cells infected with the ORF P++ virus, there was a large increase in the amount of ORF P RNA and a corresponding decrease in the amount of gamma 1 34.5 RNA. We conclude that either the overproduction of ORF P protein blocks the expression of some herpes simplex virus 1 genes or derepression of the transcription of ORF P has a negative effect on the transcription of the antisense gamma 1 34.5 RNA. PMID:8627705

  15. Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems

    PubMed Central

    Ryabova, Lyubov A.; Hohn, Thomas

    2000-01-01

    The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning–reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5′-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5′-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of reinitiation. PMID:10766738

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

    PubMed

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

    2014-09-01

    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 (6552nt) 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 (5945nt), 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. PMID:25117897

  17. Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

    PubMed Central

    Schepetilnikov, Mikhail; Schott, Gregory; Katsarou, Konstantina; Thiébeauld, Odon; Keller, Mario; Ryabova, Lyubov A.

    2009-01-01

    The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs. PMID:19638424

  18. Interaction between ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex Is Essential for Viral Late Gene Expression

    PubMed Central

    Davis, Zoe H.; Hesser, Charles R.; Park, Jimin

    2015-01-01

    Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in gammaherpesviruses requires the coordination of six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus. Disruption of this complex via point mutation of the interaction interface between the open reading frame 24 (ORF24) and ORF34 vTFs ablated both late gene expression and viral replication. PMID:26468530

  19. Avian Nephritis Virus (ANV) as a New Member of the Family Astroviridae and Construction of Infectious ANV cDNA

    PubMed Central

    Imada, Tadao; Yamaguchi, Shigeo; Mase, Masaji; Tsukamoto, Kenji; Kubo, Masanori; Morooka, Akira

    2000-01-01

    The complete RNA genome of the avian nephritis virus (ANV) associated with acute nephritis in chickens has been molecularly cloned and sequenced. Excluding the poly(A) tail, the genome comprises 6,927 nucleotides and contains three sequential open reading frames (ORFs). The first ORF (ORF 1a) contains a sequence encoding a serine protease motif, and the second ORF (ORF 1b) has a sequence encoding an RNA-dependent RNA polymerase. ORF 1a may be linked to the second ORF by a ribosomal frameshifting mechanism. The third ORF (ORF 2) may encode the virion structural proteins as a polyprotein precursor. Two RNAs, probably genonic and subgenonic RNA (7.5 and 3.0 kb), were detected in the cytoplasm of ANV-infected cells. ANV and human astroviruses have the same genonic organization, and both are characterized by the presence of two RNA bands. The amino acid homologies of the products of ORF 1a, 1b, and 2 were 20.3, 41.9, and 25.8% to products of the corresponding ORFs of human astrovirus serotype 1 (A/88 Newcastle strain). We have constructed a genonic-length cDNA clone of ANV to test whether the in vitro transcript is infectious. When a chicken kidney cell culture was transfected with RNA transcribed in vitro and the cDNA clone, infectious virus was produced with cytopathic effects in the absence of trypsin. These observations suggested that the ANV (G-4260 strain) is a new genus of the family Astroviridae. PMID:10954549

  20. Plants transformed with a region of the 201-kilodalton replicase gene from pea early browning virus RNA1 are resistant to virus infection.

    PubMed

    MacFarlane, S A; Davies, J W

    1992-07-01

    The 3' proximal portion of the gene encoding the 201-kDa putative replicase protein from the Tobravirus pea early browning virus (PEBV) can potentially be expressed separately as a 54-kDa protein. Nicotiana benthamiana plants transformed with the open reading frame (ORF) encoding the 54-kDa protein, designated 54K ORF, were resistant to infection by purified PEBV at inoculum doses of up to 1 mg/ml, the highest concentration tested. However, resistance was abolished by the introduction into the 54K ORF of mutations that would cause premature termination of translation. This suggests that the resistance mechanism requires the involvement of an intact 54-kDa protein. The 54K ORF-transformed plants were also resistant to infection by broad bean yellow band virus and an uncharacterized isolate of British PEBV (PGRO R) but were not resistant to infection by two other tobraviruses, pepper ringspot virus and the I6 isolate of tobacco rattle virus. Additionally, two variants of PEBV which overcame 54K ORF-mediated resistance have been isolated, the analysis of which might provide important information about both the resistance mechanism itself and the process of normal virus replication. PMID:1631064

  1. OrfPredictor: predicting protein-coding regions in EST-derived sequences.

    PubMed

    Min, Xiang Jia; Butler, Gregory; Storms, Reginald; Tsang, Adrian

    2005-07-01

    OrfPredictor is a web server designed for identifying protein-coding regions in expressed sequence tag (EST)-derived sequences. For query sequences with a hit in BLASTX, the program predicts the coding regions based on the translation reading frames identified in BLASTX alignments, otherwise, it predicts the most probable coding region based on the intrinsic signals of the query sequences. The output is the predicted peptide sequences in the FASTA format, and a definition line that includes the query ID, the translation reading frame and the nucleotide positions where the coding region begins and ends. OrfPredictor facilitates the annotation of EST-derived sequences, particularly, for large-scale EST projects. OrfPredictor is available at https://fungalgenome.concordia.ca/tools/OrfPredictor.html. PMID:15980561

  2. Nucleotide sequence of Zygosaccharomyces bailii virus Z: Evidence for +1 programmed ribosomal frameshifting and for assignment to family Amalgaviridae.

    PubMed

    Depierreux, Delphine; Vong, Minh; Nibert, Max L

    2016-06-01

    Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1-ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed. PMID:26951859

  3. Altered network connectivity in frontotemporal dementia with C9orf72 hexanucleotide repeat expansion.

    PubMed

    Lee, Suzee E; Khazenzon, Anna M; Trujillo, Andrew J; Guo, Christine C; Yokoyama, Jennifer S; Sha, Sharon J; Takada, Leonel T; Karydas, Anna M; Block, Nikolas R; Coppola, Giovanni; Pribadi, Mochtar; Geschwind, Daniel H; Rademakers, Rosa; Fong, Jamie C; Weiner, Michael W; Boxer, Adam L; Kramer, Joel H; Rosen, Howard J; Miller, Bruce L; Seeley, William W

    2014-11-01

    Hexanucleotide repeat expansion in C9orf72 represents the most common genetic cause of familial and sporadic behavioural variant frontotemporal dementia. Previous studies show that some C9orf72 carriers with behavioural variant frontotemporal dementia exhibit distinctive atrophy patterns whereas others show mild or undetectable atrophy despite severe behavioural impairment. To explore this observation, we examined intrinsic connectivity network integrity in patients with or without the C9orf72 expansion. We studied 28 patients with behavioural variant frontotemporal dementia, including 14 C9orf72 mutation carriers (age 58.3 ± 7.7 years, four females) and 14 non-carriers (age 60.8 ± 6.9 years, four females), and 14 age- and sex-matched healthy controls. Both patient groups included five patients with comorbid motor neuron disease. Neuropsychological data, structural brain magnetic resonance imaging, and task-free functional magnetic resonance imaging were obtained. Voxel-based morphometry delineated atrophy patterns, and seed-based intrinsic connectivity analyses enabled group comparisons of the salience, sensorimotor, and default mode networks. Single-patient analyses were used to explore network imaging as a potential biomarker. Despite contrasting atrophy patterns in C9orf72 carriers versus non-carriers, patient groups showed topographically similar connectivity reductions in the salience and sensorimotor networks. Patients without C9orf72 expansions exhibited increases in default mode network connectivity compared to controls and mutation carriers. Across all patients, behavioural symptom severity correlated with diminished salience network connectivity and heightened default mode network connectivity. In C9orf72 carriers, salience network connectivity reduction correlated with atrophy in the left medial pulvinar thalamic nucleus, and this region further showed diminished connectivity with key salience network hubs. Single-patient analyses revealed salience

  4. Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses.

    PubMed

    Stadejek, T; Stankevicius, A; Storgaard, T; Oleksiewicz, M B; Belák, S; Drew, T W; Pejsak, Z

    2002-08-01

    We determined 22 partial porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences, representing pathogenic field strains mainly from Poland and Lithuania, and two currently available European-type live PRRSV vaccines. Also, the complete ORF7 of two Lithuanian and two Polish strains was sequenced. We found that Polish, and in particular Lithuanian, PRRSV sequences were exceptionally different from the European prototype, the Lelystad virus, and in addition showed a very high national diversity. The most diverse present-day European-type PRRSV sequences were from Poland (2000) and Lithuania (2000), and exhibited only 72.2% nucleotide identity in the investigated ORF5 sequence. While all sequences determined in the present study were clearly of European type, inclusion of the new Lithuanian sequences in the genealogy resulted in a common ancestor for the European type virus significantly closer to the American-type PRRSV than previously seen. In addition, the length of the ORF7 of the Lithuanian strains was 378 nucleotides, and thus intermediate between the sizes of the prototypical EU-type (387 nucleotides) and US-type (372 nucleotides) ORF7 lengths. These findings for the Lithuanian PRRSV sequences provide support for the hypothesis that the EU and US genotypes of PRRSV evolved from a common ancestor. Also, this is the first report of ORF7 protein size polymorphism in field isolates of EU-type PRRSV. PMID:12124450

  5. Recent advances in Hepatitis E virus.

    PubMed

    Meng, X J

    2010-03-01

    Hepatitis E virus (HEV), the causative agent of hepatitis E, belongs to the family Hepeviridae. At least four major genotypes of HEV have been recognized: genotypes 1 and 2 are restricted to humans and associated with epidemics in developing countries, whereas genotypes 3 and 4 are zoonotic and infect humans and several other animals in both developing and industrialized countries. Besides humans, strains of HEV have been genetically identified from swine, chickens, sika deer, mongeese, and rabbits. The genome of HEV consists of three open reading frames (ORFs): ORF1 codes for nonstructural proteins, ORF2 codes for capsid protein, and ORF3 codes for a small multifunctional protein. The ORF2 and ORF3 proteins are translated from a single bicistronic mRNA and overlap each other but neither overlaps ORF1. The recent determination of the 3D crystal structure of the HEV capsid protein should facilitate the development of vaccines and antivirals. The identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by swine HEV raise public health concerns for zoonosis. Accumulating evidence indicated that hepatitis E is a zoonotic disease and pigs and more likely other animal species are reservoirs for HEV. This article provides an overview of the recent advances in hepatitis E and its causative agent, including nomenclature and genomic organization, gene expression and functions, 3D structure of the virions, changing perspectives on higher mortality during pregnancy and chronic hepatitis E, animal reservoirs, zoonotic risk, food safety, and novel animal models. PMID:20040046

  6. 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

  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. Molecular epidemiology of PRRSV in South China from 2007 to 2011 based on the genetic analysis of ORF5.

    PubMed

    Xie, Jiexiong; Zhu, Wanjun; Chen, Ye; Wei, Chunya; Zhou, Pei; Zhang, Minze; Huang, Zhen; Sun, Long; Su, Shuo; Zhang, Guihong

    2013-10-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has proven to be highly genetically variable; however, comprehensive information regarding the virus's genetic diversity in South China is limited. In this study, a total of 3199 clinical samples were collected from 267 pig farms suspected of PRRSV infection between 2007 and 2011. The ORF5 genes of 51 PRRSV-positive samples were sequenced and analyzed. The 51 study strains were divided into three primary subgenotypes. Fourty-five of the strains belonged to subgenotype I and were closely related to the highly pathogenic PRRSV (HP-PRRSV) strains. The subgenotype I strains were generally clustered into genetically similar groups by year. Only one of the strains belonged to subgenotype II, clustering with the classical North American type, VR2332. Five of the strains were grouped into subgenotype III, which occupied a separate branch and was closely related to the recently isolated novel field strains, QYYZ and GM2. The 5 subgenotype III strains shared an amino acid identity with the remaining 46 study strains ranging from 79.6%-83.6%. Amino acid analysis showed extensive mutations in subgenotype III; the diverse genetic mutations of these novel strains are of great concern. PMID:23770054

  9. 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

  10. 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. PMID:26832642

  11. 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

  12. 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

  13. Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells.

    PubMed

    Zhu, L; Wang, R; Sweat, A; Goldstein, E; Horvat, R; Chandran, B

    1999-04-10

    The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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

  15. 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

  16. 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

  17. 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

  18. Eimeria tenella: a novel dsRNA virus in E. tenella and its complete genome sequence analysis.

    PubMed

    Wu, Bin; Zhang, Xichen; Gong, Pengtao; Li, Mingying; Ding, He; Xin, Caiyan; Zhao, Na; Li, Jianhua

    2016-04-01

    Protozoa double-stranded (ds) RNA viruses have been described in Trichomonas, Giardia, and Leishmania. In this study, dsRNA and virus-like particles (approximately 30 nm in diameter) were discovered in Eimeria tenella sporulated oocysts. The complete genome of this novel dsRNA virus was sequenced using a three-step strategy. The sequencing results showed that the complete genome sequence was 6006 bp containing two open reading frames (ORFs) (2367 bp for ORF1 and 3216 bp for ORF2) with a five-nucleotide overlap (UGA/UG). The predicted ORF1 and ORF2 encoded a putative capsid protein of 788 amino acids (84.922 kDa) and a putative RNA-dependent RNA polymerase (RdRp) protein of 1071 amino acids (118.190 kDa). BLASTp analysis showed that the amino acid sequences for the E. tenella virus shared similarity with the E. brunetti RNA virus, with 29% homology in capsid proteins and 36% in RDRP proteins. The two untranslated regions were 349 bp (5' UTR) and 78 bp (3' UTR). The complete genome sequence of the E. tenella virus resembled characteristics of the Totiviridae family, indicating that this virus was a novel member of Totiviridae. Surprisingly, phylogenetic analysis showed that the E. tenella virus, E. brunetti RNA virus 1, and Mycoviruses were clustered into the genus Victorivirus and separated from the reported protozoa viruses, strongly suggesting a novel Eimeriaviruses subgenus. To the best of our knowledge, this is the first report for the complete genome sequence of the E. tenella virus. Using the nomenclature generally adopted for viruses, this new isolate was named E. tenella RNA virus 1. This study provides a foundation basis for further research on the biological characteristics of Eimeriaviruses. PMID:26873407

  19. 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

  20. 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.

  1. 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

  2. Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice.

    PubMed

    Kazama, Tomohiko; Itabashi, Etsuko; Fujii, Shinya; Nakamura, Takahiro; Toriyama, Kinya

    2016-03-01

    Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear-encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)-type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD-CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS-associated gene in LD-CMS rice, similar to its role in BT-CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT-CMS rice. We also show that RF2 promotes degradation of atp6-orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT-CMS rice. The amount of ORF79 protein in LD-CMS rice was one-twentieth of the amount in BT-CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD-CMS and BT-CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD-CMS and BT-CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD-CMS and BT-CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79. PMID:26850149

  3. A Mutation in C2orf64 Causes Impaired Cytochrome c Oxidase Assembly and Mitochondrial Cardiomyopathy

    PubMed Central

    Huigsloot, Merei; Nijtmans, Leo G.; Szklarczyk, Radek; Baars, Marieke J.H.; van den Brand, Mariël A.M.; HendriksFranssen, Marthe G.M.; van den Heuvel, Lambertus P.; Smeitink, Jan A.M.; Huynen, Martijn A.; Rodenburg, Richard J.T.

    2011-01-01

    The assembly of mitochondrial respiratory chain complex IV (cytochrome c oxidase) involves the coordinated action of several assembly chaperones. In Saccharomyces cerevisiae, at least 30 different assembly chaperones have been identified. To date, pathogenic mutations leading to a mitochondrial disorder have been identified in only seven of the corresponding human genes. One of the genes for which the relevance to human pathology is unknown is C2orf64, an ortholog of the S. cerevisiae gene PET191. This gene has previously been shown to be a complex IV assembly factor in yeast, although its exact role is still unknown. Previous research in a large cohort of complex IV deficient patients did not support an etiological role of C2orf64 in complex IV deficiency. In this report, a homozygous mutation in C2orf64 is described in two siblings affected by fatal neonatal cardiomyopathy. Pathogenicity of the mutation is supported by the results of a complementation experiment, showing that complex IV activity can be fully restored by retroviral transduction of wild-type C2orf64 in patient-derived fibroblasts. Detailed analysis of complex IV assembly intermediates in patient fibroblasts by 2D-BN PAGE revealed the accumulation of a small assembly intermediate containing subunit COX1 but not the COX2, COX4, or COX5b subunits, indicating that C2orf64 is involved in an early step of the complex IV assembly process. The results of this study demonstrate that C2orf64 is essential for human complex IV assembly and that C2orf64 mutational analysis should be considered for complex IV deficient patients, in particular those with hypertrophic cardiomyopathy. PMID:21457908

  4. A novel single-stranded RNA virus isolated from the rice-pathogenic fungus Magnaporthe oryzae with similarity to members of the family Tombusviridae.

    PubMed

    Ai, Ye-Ping; Zhong, Jie; Chen, Chuan-Yuan; Zhu, Hong-Jian; Gao, Bi-Da

    2016-03-01

    Here, we report a novel virus isolated from rice blast fungus, Magnaporthe oryzae, an important plant pathogen. This virus has an RNA genome of 3246 nucleotides. Its genome possesses two in-frame open reading frames (ORFs). The smaller ORF1 encodes a protein with significant similarity to a protein encoded by the ssRNA mycovirus Diaporthe ambigua RNA virus 1 (DaRV1). The larger ORF2 encodes a protein with similarity to RNA-dependent RNA polymerases (RdRp) of DaRV1 and other plant viruses of the family Tombusviridae. In silico analysis and comparisons with DaRV1 genome expression suggest that ORF2 is translated via a readthrough mechanism together with ORF1. Based upon results of this study, this virus, for which the provisional name Magnaporthe oryzae virus A (MoVA) is proposed, belongs to a new virus species. Furthermore, MoVA along with DaRV1 belong to a new taxon of mycoviruses that are evolutionarily related to plant viruses belonging to the family Tombusviridae. PMID:26650038

  5. 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 Central

    Gunišová, Stanislava; Valášek, Leoš Shivaya

    2014-01-01

    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. PMID:24623812

  6. 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

  7. 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

  8. 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

  9. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    PubMed

    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

  10. Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola.

    PubMed

    Zhong, Jie; Shang, Hong Hong; Zhu, Chuan Xia; Zhu, Jun Zi; Zhu, Hong Jian; Hu, Yan; Gao, Bi Da

    2016-06-01

    The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host. PMID:26707921

  11. Impairment of Drosophila Orthologs of the Human Orphan Protein C19orf12 Induces Bang Sensitivity and Neurodegeneration

    PubMed Central

    Iuso, Arcangela; Sibon, Ody C. M.; Gorza, Matteo; Heim, Katharina; Organisti, Cristina; Meitinger, Thomas; Prokisch, Holger

    2014-01-01

    Mutations in the orphan gene C19orf12 were identified as a genetic cause in a subgroup of patients with NBIA, a neurodegenerative disorder characterized by deposits of iron in the basal ganglia. C19orf12 was shown to be localized in mitochondria, however, nothing is known about its activity and no functional link exists to the clinical phenotype of the patients. This situation led us to investigate the effects of C19orf12 down-regulation in the model organism Drosophila melanogaster. Two genes are present in D. melanogaster, which are orthologs of C19orf12, CG3740 and CG11671. Here we provide evidence that transgenic flies with impaired C19orf12 homologs reflect the neurodegenerative phenotype and represent a valid tool to further analyze the pathomechanism in C19orf12-associated NBIA. PMID:24586779

  12. Deletion of Murid Herpesvirus 4 ORF63 Affects the Trafficking of Incoming Capsids toward the Nucleus

    PubMed Central

    Latif, Muhammad Bilal; Machiels, Bénédicte; Xiao, Xue; Mast, Jan; Vanderplasschen, Alain

    2015-01-01

    ABSTRACT Gammaherpesviruses are important human and animal pathogens. Despite the fact that they display the classical architecture of herpesviruses, the function of most of their structural proteins is still poorly defined. This is especially true for tegument proteins. Interestingly, a potential role in immune evasion has recently been proposed for the tegument protein encoded by Kaposi's sarcoma-associated herpesvirus open reading frame 63 (ORF63). To gain insight about the roles of ORF63 in the life cycle of a gammaherpesvirus, we generated null mutations in the ORF63 gene of murid herpesvirus 4 (MuHV-4). We showed that disruption of ORF63 was associated with a severe MuHV-4 growth deficit both in vitro and in vivo. The latter deficit was mainly associated with a defect of replication in the lung but did not affect the establishment of latency in the spleen. From a functional point of view, inhibition of caspase-1 or the inflammasome did not restore the growth of the ORF63-deficient mutant, suggesting that the observed deficit was not associated with the immune evasion mechanism identified previously. Moreover, this growth deficit was also not associated with a defect in virion egress from the infected cells. In contrast, it appeared that MuHV-4 ORF63-deficient mutants failed to address most of their capsids to the nucleus during entry into the host cell, suggesting that ORF63 plays a role in capsid movement. In the future, ORF63 could therefore be considered a target to block gammaherpesvirus infection at a very early stage of the infection. IMPORTANCE The important diseases caused by gammaherpesviruses in human and animal populations justify a better understanding of their life cycle. In particular, the role of most of their tegument proteins is still largely unknown. In this study, we used murid herpesvirus 4, a gammaherpesvirus infecting mice, to decipher the role of the protein encoded by the viral ORF63 gene. We showed that the absence of this protein is

  13. 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

  14. 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

  15. 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

  16. Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

    PubMed

    Klute, K A; Nadler, S A; Stenger, D C

    1996-07-01

    The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed. PMID:8757976

  17. Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells.

    PubMed

    Černý, Jiří; Selinger, Martin; Palus, Martin; Vavrušková, Zuzana; Tykalová, Hana; Bell-Sakyi, Lesley; Štěrba, Ján; Grubhoffer, Libor; Růžek, Daniel

    2016-06-01

    A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle. PMID:26924586

  18. 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

  19. Oral-facial-digital syndrome type VI: is C5orf42 really the major gene?

    PubMed

    Romani, Marta; Mancini, Francesca; Micalizzi, Alessia; Poretti, Andrea; Miccinilli, Elide; Accorsi, Patrizia; Avola, Emanuela; Bertini, Enrico; Borgatti, Renato; Romaniello, Romina; Ceylaner, Serdar; Coppola, Giangennaro; D'Arrigo, Stefano; Giordano, Lucio; Janecke, Andreas R; Lituania, Mario; Ludwig, Kathrin; Martorell, Loreto; Mazza, Tommaso; Odent, Sylvie; Pinelli, Lorenzo; Poo, Pilar; Santucci, Margherita; Signorini, Sabrina; Simonati, Alessandro; Spiegel, Ronen; Stanzial, Franco; Steinlin, Maja; Tabarki, Brahim; Wolf, Nicole I; Zibordi, Federica; Boltshauser, Eugen; Valente, Enza Maria

    2015-01-01

    Oral-facial-digital type VI syndrome (OFDVI) is a rare phenotype of Joubert syndrome (JS). Recently, C5orf42 was suggested as the major OFDVI gene, being mutated in 9 of 11 families (82 %). We sequenced C5orf42 in 313 JS probands and identified mutations in 28 (8.9 %), most with a phenotype of pure JS. Only 2 out of 17 OFDVI patients (11.7 %) were mutated. A comparison of mutated vs. non-mutated OFDVI patients showed that preaxial and mesoaxial polydactyly, hypothalamic hamartoma and other congenital defects may predict C5orf42 mutations, while tongue hamartomas are more common in negative patients. PMID:25407461

  20. 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

  1. 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

  2. Donkey Orchid Symptomless Virus: A Viral ‘Platypus’ from Australian Terrestrial Orchids

    PubMed Central

    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

  3. Forced Evolution Reveals the Importance of Short Open Reading Frame A and Secondary Structure in the Cauliflower Mosaic Virus 35S RNA Leader

    PubMed Central

    Pooggin, Mikhail M.; Hohn, Thomas; Fütterer, Johannes

    1998-01-01

    Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5′-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed. PMID:9557705

  4. 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

  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. Activation of p90 Ribosomal S6 Kinase by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus and Its Role in Viral Lytic Replication▿

    PubMed Central

    Kuang, Ersheng; Tang, Qiyi; Maul, Gerd G.; Zhu, Fanxiu

    2008-01-01

    The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication. PMID:18057234

  7. 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

  8. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits.

    PubMed

    Chew, Jeannie; Gendron, Tania F; Prudencio, Mercedes; Sasaguri, Hiroki; Zhang, Yong-Jie; Castanedes-Casey, Monica; Lee, Chris W; Jansen-West, Karen; Kurti, Aishe; Murray, Melissa E; Bieniek, Kevin F; Bauer, Peter O; Whitelaw, Ena C; Rousseau, Linda; Stankowski, Jeannette N; Stetler, Caroline; Daughrity, Lillian M; Perkerson, Emilie A; Desaro, Pamela; Johnston, Amelia; Overstreet, Karen; Edbauer, Dieter; Rademakers, Rosa; Boylan, Kevin B; Dickson, Dennis W; Fryer, John D; Petrucelli, Leonard

    2015-06-01

    The major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis is a G4C2 repeat expansion in C9ORF72. Efforts to combat neurodegeneration associated with "c9FTD/ALS" are hindered by a lack of animal models recapitulating disease features. We developed a mouse model to mimic both neuropathological and clinical c9FTD/ALS phenotypes. We expressed (G4C2)66 throughout the murine central nervous system by means of somatic brain transgenesis mediated by adeno-associated virus. Brains of 6-month-old mice contained nuclear RNA foci, inclusions of poly(Gly-Pro), poly(Gly-Ala), and poly(Gly-Arg) dipeptide repeat proteins, as well as TDP-43 pathology. These mouse brains also exhibited cortical neuron and cerebellar Purkinje cell loss, astrogliosis, and decreased weight. (G4C2)66 mice also developed behavioral abnormalities similar to clinical symptoms of c9FTD/ALS patients, including hyperactivity, anxiety, antisocial behavior, and motor deficits. PMID:25977373

  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. 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

  11. An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome.

    PubMed

    Blanchard, Ph; Mahé, D; Cariolet, R; Truong, C; Le Dimna, M; Arnauld, C; Rose, N; Eveno, E; Albina, E; Madec, F; Jestin, A

    2003-07-17

    Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets. PMID:12814886

  12. 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.

  13. 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

  14. 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. PMID:3671088

  15. Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

    PubMed Central

    Kitano, Shouta; Kino, Yoshihiro; Yamamoto, Yoji; Takitani, Mika; Miyoshi, Junko; Ishida, Tsuyoshi; Saito, Yuko; Arima, Kunimasa; Satoh, Jun-ichi

    2015-01-01

    BACKGROUND Expanded GGGGCC hexanucleotide repeats located in the noncoding region of the chromosome 9 open reading frame 72 (C9orf72) gene represent the most common genetic abnormality for familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Formation of nuclear RNA foci, accumulation of repeat-associated non-ATG-translated dipeptide-repeat proteins, and haploinsufficiency of C9orf72 are proposed for pathological mechanisms of C9ALS/FTD. However, at present, the physiological function of C9orf72 remains largely unknown. METHODS By searching on a bioinformatics database named COXPRESdb composed of the comprehensive gene coexpression data, we studied potential C9orf72 interactors. RESULTS We identified the ATP/GTP binding protein 1 (AGTPBP1) gene alternatively named NNA1 encoding a cytosolic carboxypeptidase whose mutation is causative of the degeneration of Purkinje cells and motor neurons as the most significant gene coexpressed with C9orf72. We verified coexpression and interaction of AGTPBP1 and C9orf72 in transfected cells by immunoprecipitation and in neurons of the human brain by double-labeling immunohistochemistry. Furthermore, we found a positive correlation between AGTPBP1 and C9orf72 mRNA expression levels in the set of 21 human brains examined. CONCLUSIONS These results suggest that AGTPBP1 serves as a C9orf72 interacting partner that plays a role in the regulation of neuronal function in a coordinated manner within the central nervous system. PMID:26106267

  16. 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

  17. Association of epigenetic alterations in the human C7orf24 gene with the aberrant gene expression in malignant cells.

    PubMed

    Ohno, Yuji; Hattori, Akira; Yoshiki, Tatsuhiro; Kakeya, Hideaki

    2013-10-01

    Human chromosome 7 open reading frame 24 (C7orf24)/γ-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics. PMID:23853312

  18. C9orf72 promoter hypermethylation is reduced while hydroxymethylation is acquired during reprogramming of ALS patient cells.

    PubMed

    Esanov, Rustam; Belle, Kinsley C; van Blitterswijk, Marka; Belzil, Veronique V; Rademakers, Rosa; Dickson, Dennis W; Petrucelli, Leonard; Boylan, Kevin B; Dykxhoorn, Derek M; Wuu, Joanne; Benatar, Michael; Wahlestedt, Claes; Zeier, Zane

    2016-03-01

    Among several genetic mutations known to cause amyotrophic lateral sclerosis (ALS), a hexanucleotide repeat expansion in the C9orf72 gene is the most common. In approximately 30% of C9orf72-ALS cases, 5-methylcytosine (5mC) levels within the C9orf72 promoter are increased, resulting in a modestly attenuated phenotype. The developmental timing of C9orf72 promoter hypermethylation and the reason why it occurs in only a subset of patients remain unknown. In order to model the acquisition of C9orf72 hypermethylation and examine the potential role of 5-hydroxymethylcytosine (5hmC), we generated induced pluripotent stem cells (iPSCs) from an ALS patient with C9orf72 promoter hypermethylation. Our data show that 5mC levels are reduced by reprogramming and then re-acquired upon neuronal specification, while 5hmC levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of 5hmC within the C9orf72 promoter in post-mortem brain tissues of hypermethylated patients. These findings show that iPSCs are a valuable model system for examining epigenetic perturbations caused by the C9orf72 mutation and reveal a potential role for cytosine demethylation. PMID:26746986

  19. C9ORF72 Intermediate Repeat Copies Are a Significant Risk Factor for Parkinson Disease

    PubMed Central

    Nuytemans, Karen; Bademci, Güney; Kohli, Martin M; Beecham, Gary W; Wang, Liyong; Young, Juan I; Nahab, Fatta; Martin, Eden R; Gilbert, John R; Benatar, Michael; Haines, Jonathan L; Scott, William K; Züchner, Stephan; Pericak-Vance, Margaret A; Vance, Jeffery M

    2013-01-01

    We set out to determine whether expansions in the C9ORF72 repeat found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) families are associated with Parkinson disease (PD). We determined the repeat size in a total of 889 clinically ascertained patients (including PD and essential tremor plus Parkinsonism (ETP)) and 1144 controls using a repeat-primed PCR assay. We found that large C9ORF72 repeat expansions (>30 repeats) were not contributing to PD risk. However, PD and ETP cases had a significant increase in intermediate (>20 to 30+) repeat copies compared to controls. Overall, 14 cases (13 PD, 1 ETP) and three controls had >20 repeat copies (Fisher's exact test p = 0.002). Further, seven cases and no controls had >23 repeat copies (p = 0.003). Our results suggest that intermediate copy numbers of the C9ORF72 repeat contribute to risk for PD and ETP. This also suggests that PD, ALS and FTD share some pathophysiological mechanisms of disease. Further studies are needed to elucidate the contribution of the C9ORF72 repeat in the overall PD population and to determine whether other common genetic risk factors exist between these neurodegenerative disorders. PMID:23845100

  20. C11ORF95-RELA FUSIONS DRIVE ONCOGENIC NF-KB SIGNALING IN EPENDYMOMA

    PubMed Central

    Gilbertson, Richard; Parker, Matthew; Mohankumar, Kumarasamypet M.; Punchihewa, Chandanamali; Weinlich, Ricardo; Dalton, James D.; Li, Yongjin; Lee, Ryan; Tatevossian, Ruth G.; Phoenix, Timothy N.; Thiruvenkatam, Radhika; White, Elsie; Tang, Bo; Orisme, Wilda; Gupta, Kirti; Rusch, Michael; Chen, Xiang; Li, Yuxin; Nagahawhatta, Panduka; Hedlund, Erin; Finkelstein, David; Wu, Gang; Shurtleff, Sheila; Easton, John; Boggs, Kristy; Yergeau, Donald; Vadodaria, Bhavin; Mulder, Heather L.; Becksford, Jared; Gupta, Pankaj; Huether, Robert; Ma, Jing; Song, Guangchun; Gajjar, Amar; Merchant, Thomas; Boop, Frederick; Smith, Amy A; Ding, Li; Lu, Charles; Ochoa, Kerri; Zhao, David; Fulton, Robert S.; Fulton, Lucinda L.; Mardis, Elaine R.; Wilson, Richard K.; Downing, James R.; Green, Douglas R.; Zhang, Jinghui; Ellison, David W.; Gilbertson, Richard J.

    2014-01-01

    BACKGROUND: The nuclear factor-kB (NF-kB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-kB signaling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-kB activity in cancer. METHODS: To identify additional genetic alterations that drive ependymoma, we sequenced the whole genomes (WGS) of 41 tumours and matched normal blood, and the transcriptomes (RNAseq) of 77 tumours. The transforming significance of alterations were tested in mouse NSCs that we showed previously to be cells of origin of ependymoma. RESULTS: Here, we show that more than two thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-kB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-kB target genes, and rapidly transformed neural stem cells—the cell of origin of ependymoma—to form these tumours in mice. CONCLUSIONS: Our data identify the first highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

  1. Genetic counseling for FTD/ALS caused by the C9ORF72 hexanucleotide expansion

    PubMed Central

    2012-01-01

    Frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) are related but distinct neurodegenerative diseases. The identification of a hexanucleotide repeat expansion in a noncoding region of the chromosome 9 open reading frame 72 (C9ORF72) gene as a common cause of FTD/ALS, familial FTD, and familial ALS marks the culmination of many years of investigation. This confirms the linkage of disease to chromosome 9 in large, multigenerational families with FTD and ALS, and it promotes deeper understanding of the diseases' shared molecular FTLD-TDP pathology. The discovery of the C9ORF72 repeat expansion has significant implications not only for familial FTD and ALS, but also for sporadic disease. Clinical and pathological correlates of the repeat expansion are being reported but remain to be refined, and a genetic test to detect the expansion has only recently become clinically available. Consequently, individuals and their families who are considering genetic testing for the C9ORF72 expansion should receive genetic counseling to discuss the risks, benefits, and limitations of testing. The following review aims to describe genetic counseling considerations for individuals at risk for a C9ORF72 repeat expansion. PMID:22808918

  2. C9orf72 associated FTD/ALS: When less is more

    PubMed Central

    Todd, Peter K.; Paulson, Henry L.

    2016-01-01

    Hexanucleotide repeat expansions in C9ORF72 cause neurodegeneration in FTD and ALS by unknown mechanisms. A new report finds that these repeats trigger a pathogenic gain-of-function cascade that can be corrected by suppressing expression of the repeat transcript, paving the way for therapeutic strategies aimed at eliminating the toxic RNA. PMID:24139028

  3. C9ORF72-ALS/FTD: Transgenic Mice Make a Come-BAC.

    PubMed

    Hayes, Lindsey R; Rothstein, Jeffrey D

    2016-05-01

    For five years, since the landmark discovery of the C9ORF72 hexanucleotide repeat expansion in ALS/FTD, a transgenic mouse model has remained elusive. Now, two laboratories (Liu et al., 2016; Jiang et al., 2016) report the development of BAC transgenic mice that recapitulate features of the human disease. PMID:27151634

  4. Analysis of the C9orf72 gene in spinal muscular atrophy patients.

    PubMed

    Alías, Laura; Bernal, Sara; Barceló, Maria J; Martínez-Hernández, Rebeca; Martínez, Elisabeth; Baiget, Montserrat; Tizzano, Eduardo F

    2014-12-01

    Spinal muscular atrophy and amyotrophic lateral sclerosis are both motor neuron disorders. Several studies have tried to establish a link between the two diseases but the subject is still under debate. In amyotrophic lateral sclerosis, large expansions of the hexanucleotide GGGGCC in intron 1 of the C9orf72 gene are responsible for a variable percentage of familial and sporadic cases. We investigated whether the number of the hexanucleotide repeat in C9orf72 was associated with the phenotype and the number of SMN2 copies in a group of 162 SMA patients. Conventional PCR, repeat primed-PCR and Southern blot were used to determine repeat number and characterize large expansions. Results showed that no pathological (> 30 repeats) or premutated alleles (20-30 repeats) were found. The allelic distribution of the C9orf72 gene in spinal muscular atrophy patients overlapped with the data obtained in our control population, discarding putative repeats that may be associated with the disease. No association was observed with either the SMA phenotype or the number of SMN2 copies. In conclusion, the involvement of C9orf72 as a genetic modifier in spinal muscular atrophy is unlikely. Current investigation of modifier genes in SMA and of the link between ALS and SMA should consider other possible candidates. PMID:24998634

  5. Photosensitivity in a patient with C9orf72 repeat expansion.

    PubMed

    Janssen, Paula; Houben, Mark; Hoff, Erik

    2016-01-01

    The phenotype of C9ORF72 repeat expansions is rapidly expanding. Originally found to be the major genetic cause of familial frontotemporal dementia with amyotrophic lateral sclerosis, several other clinical characteristics have been described more recently. Here, we report on a family diagnosed with 'degenerative schizophrenia' and harbouring a C9ORF72 repeat expansion, in which at least the index patient and an uncle showed a photoparoxysmal response during electroencephalography. Whereas epilepsy has been described in several repeat expansion disorders, photosensitivity has thus far only been reported in dentatorubral-pallidoluysian atrophy. The photoparoxysmal response may therefore be a new clinical feature of C9ORF72 repeat expansion related disease. Our observation learns that a repeat expansion disorder like C9ORF72 should be considered in patients with a combination of young-onset dementia, psychiatric symptoms and/or photosensitive epilepsy. We advocate the occasional use of EEG in the dementia workup and in particular urge to consider provocative tests such as photic stimulation. PMID:26735706

  6. The C20orf133 gene is disrupted in a patient with Kabuki syndrome

    PubMed Central

    Maas, Nicole M C; Van de Putte, Tom; Melotte, Cindy; Francis, Annick; Schrander‐Stumpel, Constance T R M; Sanlaville, Damien; Genevieve, David; Lyonnet, Stanislas; Dimitrov, Boyan; Devriendt, Koenraad; Fryns, Jean‐Pierre; Vermeesch, Joris R

    2007-01-01

    Background Kabuki syndrome (KS) is a rare, clinically recognisable, congenital mental retardation syndrome. The aetiology of KS remains unknown. Methods Four carefully selected patients with KS were screened for chromosomal imbalances using array comparative genomic hybridisation at 1 Mb resolution. Results In one patient, a 250 kb de novo microdeletion at 20p12.1 was detected, deleting exon 5 of C20orf133. The function of this gene is unknown. In situ hybridisation with the mouse orthologue of C20orf133 showed expression mainly in brain, but also in kidney, eye, inner ear, ganglia of the peripheral nervous system and lung. Conclusion The de novo nature of the deletion, the expression data and the fact that C20orf133 carries a macro domain, suggesting a role for the gene in chromatin biology, make the gene a likely candidate to cause the phenotype in this patient with KS. Both the finding of different of chromosomal rearrangements in patients with KS features and the absence of C20orf133 mutations in 19 additional patients with KS suggest that KS is genetically heterogeneous. PMID:17586838

  7. Efficient identification of phosphatidylserine-binding proteins by ORF phage display

    SciTech Connect

    Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

    2009-08-14

    To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

  8. 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

  9. C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.

    PubMed

    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

  10. 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

  11. Gene-specific mitochondria dysfunctions in human TARDBP and C9ORF72 fibroblasts.

    PubMed

    Onesto, Elisa; Colombrita, Claudia; Gumina, Valentina; Borghi, Maria Orietta; Dusi, Sabrina; Doretti, Alberto; Fagiolari, Gigliola; Invernizzi, Federica; Moggio, Maurizio; Tiranti, Valeria; Silani, Vincenzo; Ratti, Antonia

    2016-01-01

    Dysregulation of RNA metabolism represents an important pathogenetic mechanism in both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) due to the involvement of the DNA/RNA-binding proteins TDP-43 and FUS and, more recently, of C9ORF72. A potential link between dysregulation of RNA metabolism and mitochondrial dysfunction is recently emerged in TDP-43 disease models. To further investigate the possible relationship between these two pathogenetic mechanisms in ALS/FTD, we studied mitochondria functionality in human mutant TARDBP(p.A382T) and C9ORF72 fibroblasts grown in galactose medium to induce a switch from a glycolytic to an oxidative metabolism. In this condition we observed significant changes in mitochondria morphology and ultrastructure in both mutant cells with a fragmented mitochondria network particularly evident in TARDBP(p.A382T) fibroblasts. From analysis of the mitochondrial functionality, a decrease of mitochondria membrane potential with no alterations in oxygen consumption rate emerged in TARDBP fibroblasts. Conversely, an increased oxygen consumption and mitochondria hyperpolarization were observed in C9ORF72 fibroblasts in association to increased ROS and ATP content. We found evidence of autophagy/mitophagy in dynamic equilibrium with the biogenesis of novel mitochondria, particularly in mutant C9ORF72 fibroblasts where an increase of mitochondrial DNA content and mass, and of PGC1-α protein was observed. Our imaging and biochemical data show that wild-type and mutant TDP-43 proteins do not localize at mitochondria so that the molecular mechanisms responsible for such mitochondria impairment remain to be further elucidated. For the first time our findings assess a link between C9ORF72 and mitochondria dysfunction and indicate that mitochondria functionality is affected in TARDBP and C9ORF72 fibroblasts with gene-specific features in oxidative conditions. As in neuronal metabolism mitochondria are actively used for ATP

  12. 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

  13. 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

  14. 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

  15. A novel totivirus-like virus isolated from bat guano.

    PubMed

    Yang, Xinglou; Zhang, Yunzhi; Ge, Xingyi; Yuan, Junfa; Shi, Zhengli

    2012-06-01

    Previous metagenomic analysis indicated that numerous insect viruses exist in bat guano. In this study, we isolated a novel double-stranded RNA virus, a tentative member of the family Totiviridae, designated Tianjin totivirus (ToV-TJ), from bat feces. The virus is an icosahedral particle with a diameter of 40-43 nm, and it causes cytopathic effect in Sf9, Hz, and C6/36 cell lines. Full-length genomic sequence analysis showed that ToV-TJ shares high similarity with the totivirus OMRV-AK4, which was recently isolated from mosquitoes in Japan. The full-length genome of the ToV-TJ was 7611 bp and contained two predicted non-overlapping open reading frames (ORFs): ORF1, encoding the capsid protein (CP), and ORF2, encoding an RNA-dependent RNA polymerase. Bioassay of ToV-TJ by feeding on the larvae of Spodoptera exigua and Helicoverpa armigera (Hubner) suggests that this virus is not infectious for these two larvae in vivo. Sequences similar to that of ToV-TJ have been detected in bat feces sampled in Yunnan and Hainan Provinces, suggesting that this virus is widely distributed. PMID:22421961

  16. 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.

  17. 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.

  18. Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide.

    PubMed

    Yazdi, H R Bolok; Heydarnejad, J; Massumi, H

    2008-06-01

    Beet curly top Iran virus (BCTIV) was previously reported as a distinct curtovirus in Iran. Complete nucleotide sequences of three BCTIV isolates, one each from central, southern, and south eastern Iran were determined to be 2844, 2844, and 2845 nt long, respectively. BCTIV shared highest nucleotide sequence identity (52.3%) with Spinach curly top virus (SpCTV) and lowest identity (46.6%) with Horseradish curly top virus (HrCTV). The BCTIV genome comprises three virion-sense (V1, V2, and V3) and two complementary-sense (C1 and C2) ORFs. ORFs C3 and C4 were not found in BCTIV genome. Based on a comparison of nucleotide sequence identity of individual genes, the three virion-sense ORFs were 72.7-79.9% related to the corresponding ORFs of curtoviruses, whereas no significant relationship was found between the C1 and C2 ORFs of BCTIV and curtoviruses. These two ORFs, however, were only distantly related with those of mastreviruses. Similar to the latter viruses, the BCTIV genome comprises two intergenic regions. The BCTIV large intergenic region included a sequence capable of forming a stem loop structure and a novel nonanucleotide (TAAGATT/CC) with a unique nick site. Phylogenetic analysis using deduced amino acid sequence of individual ORFs revealed that the V2 and V3 ORFs are monophyletic and the V1 ORF is classified with the related ORF of curtoviruses. Whereas the two complementary-sense ORFs are grouped with those of mastreviruses. Computer-based prediction suggested that BCTIV has a chimeric genome which may have arisen by a recombination event involving curto- and mastrevirus ancestors. Percent nucleotide sequence identities of the coat protein gene of ten isolates of BCTIV, collected from a wide range of geographical regions in Iran, varied from 87.1 to 99.9, with the isolates being distributed between two subgroups. Based on biological and molecular properties, BCTIV is proposed as a new member of the genus Curtovirus. PMID:18401697

  19. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

    PubMed

    Moreno-Vivian, C; Hennecke, S; Pühler, A; Klipp, W

    1989-05-01

    DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit. PMID:2708314

  20. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

    PubMed Central

    Moreno-Vivian, C; Hennecke, S; Pühler, A; Klipp, W

    1989-01-01

    DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit. PMID:2708314

  1. 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

  2. Molecular analysis of the complete genomic sequences of four isolates of Gooseberry vein banding associated virus.

    PubMed

    Xu, Donglin; Mock, Ray; Kinard, Gary; Li, Ruhui

    2011-08-01

    The presence of Gooseberry vein banding associated virus (GVBaV), a badnavirus in the family Caulimoviridae, is strongly correlated with gooseberry vein banding disease in Ribes spp. In this study, full-length genomic sequences of four GVBaV isolates from different hosts and geographic regions were determined to be 7649-7663 nucleotides. These isolates share identities of 96.4-97.3% for the complete genomic sequence, indicating low genetic diversity among them. The GVBaV genome contains three open reading frames (ORFs) on the plus strand that potentially encode proteins of 26, 16, and 216 kDa. The size and organization of GVBaV ORFs 1-3 are similar to those of most other badnaviruses. The putative amino acid sequence of GVBaV ORF 3 contained motifs that are conserved among badnavirus proteins including aspartic protease, reverse transcriptase, and ribonuclease H. The highly conserved putative plant tRNA(met)-binding site is also present in the 935-bp intergenic region of GVBaV. The identities of the genomic sequences of GVBaV and other badnaviruses range from 49.1% (Sugarcane bacilliform Mor virus) to 51.7% (Pelargonium vein banding virus, PVBV). Phylogenetic analysis using the amino acid sequence of the ORF 3 putative protein shows that GVBaV groups most closely to Dioscorea bacilliform virus, PVBV, and Taro bacilliform virus. These results confirm that GVBaV is a pararetrovirus of the genus Badnavirus in the family Caulimoviridae. PMID:21533750

  3. 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

  4. Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

    PubMed

    Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

    2015-07-01

    The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. PMID:25795313

  5. Enhanced Mucosal Immune Responses Induced by a Combined Candidate Mucosal Vaccine Based on Hepatitis A Virus and Hepatitis E Virus Structural Proteins Linked to Tuftsin

    PubMed Central

    Gao, Yan; Su, Qiudong; Yi, Yao; Jia, Zhiyuan; Wang, Hao; Lu, Xuexin; Qiu, Feng; Bi, Shengli

    2015-01-01

    Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368–607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1–198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection. PMID:25875115

  6. 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.

  7. 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

  8. 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

  9. 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

  10. CSBF/C10orf99, a novel potential cytokine, inhibits colon cancer cell growth through inducing G1 arrest

    PubMed Central

    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

  11. Periventricular heterotopia in 6q terminal deletion syndrome: role of the C6orf70 gene.

    PubMed

    Conti, Valerio; Carabalona, Aurelie; Pallesi-Pocachard, Emilie; Parrini, Elena; Leventer, Richard J; Buhler, Emmanuelle; McGillivray, George; Michel, François J; Striano, Pasquale; Mei, Davide; Watrin, Françoise; Lise, Stefano; Pagnamenta, Alistair T; Taylor, Jenny C; Kini, Usha; Clayton-Smith, Jill; Novara, Francesca; Zuffardi, Orsetta; Dobyns, William B; Scheffer, Ingrid E; Robertson, Stephen P; Berkovic, Samuel F; Represa, Alfonso; Keays, David A; Cardoso, Carlos; Guerrini, Renzo

    2013-11-01

    Periventricular nodular heterotopia is caused by defective neuronal migration that results in heterotopic neuronal nodules lining the lateral ventricles. Mutations in filamin A (FLNA) or ADP-ribosylation factor guanine nucleotide-exchange factor 2 (ARFGEF2) cause periventricular nodular heterotopia, but most patients with this malformation do not have a known aetiology. Using comparative genomic hybridization, we identified 12 patients with developmental brain abnormalities, variably combining periventricular nodular heterotopia, corpus callosum dysgenesis, colpocephaly, cerebellar hypoplasia and polymicrogyria, harbouring a common 1.2 Mb minimal critical deletion in 6q27. These anatomic features were mainly associated with epilepsy, ataxia and cognitive impairment. Using whole exome sequencing in 14 patients with isolated periventricular nodular heterotopia but no copy number variants, we identified one patient with periventricular nodular heterotopia, developmental delay and epilepsy and a de novo missense mutation in the chromosome 6 open reading frame 70 (C6orf70) gene, mapping in the minimal critical deleted region. Using immunohistochemistry and western blots, we demonstrated that in human cell lines, C6orf70 shows primarily a cytoplasmic vesicular puncta-like distribution and that the mutation affects its stability and subcellular distribution. We also performed in utero silencing of C6orf70 and of Phf10 and Dll1, the two additional genes mapping in the 6q27 minimal critical deleted region that are expressed in human and rodent brain. Silencing of C6orf70 in the developing rat neocortex produced periventricular nodular heterotopia that was rescued by concomitant expression of wild-type human C6orf70 protein. Silencing of the contiguous Phf10 or Dll1 genes only produced slightly delayed migration but not periventricular nodular heterotopia. The complex brain phenotype observed in the 6q terminal deletion syndrome likely results from the combined

  12. Molecular Characterization of Genome Sequences of Beak and Feather Disease Virus from the Australian Twenty-Eight Parrot (Barnardius zonarius semitorquatus).

    PubMed

    Sarker, Subir; Das, Shubhagata; Ghorashi, Seyed A; Forwood, Jade K; Raidal, Shane R

    2014-01-01

    Three complete genomes of beak and feather disease virus (BFDV) were recovered from wild twenty-eight parrots (Polytelis anthopeplus monarchoides). The genomes consisted of 1,996 bp with 1,934 identical sites and a typically content stem-loop structure between ORF1 and ORF2. This is the first report of BFDV infection as well as the complete genome sequences for this host species globally. PMID:25477410

  13. 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

  14. Can ALS-Associated C9orf72 Repeat Expansions Be Diagnosed on a Blood DNA Test Alone?

    PubMed Central

    Pamphlett, Roger; Cheong, Pak Leng; Trent, Ronald J.; Yu, Bing

    2013-01-01

    Gene mutations that preferentially affect the CNS have been implicated in a number of neurological disorders. This leads to the possibility that a disease-causing mutation present only in CNS tissues could be missed if it were tested in a blood DNA sample only. The commonest mutation in amyotrophic lateral sclerosis (ALS) is an expansion of the hexanucleotide repeats of C9orf72. To find out if CNS-specific mutations of this gene could cause some cases of ALS we looked for differences in the size of C9orf72 repeats between DNA from the CNS and from white blood cells (WBCs) of 38 sporadic ALS patients, using a repeat-primed PCR screening test. We also looked for differences in C9orf72 repeats in WBC DNA from 6 ALS-discordant and 1 ALS-concordant monozygotic twins. Abnormally expanded C9orf72 repeats were found in 13% of the ALS CNS samples, as well as in their paired WBC DNA. The 87% of ALS CNS samples with normal-sized C9orf72 repeats had the same number of repeats in paired WBC samples. All ALS-discordant twins had the same normal numbers of WBC C9orf72 repeats. Although previous work suggests some tissue mosaicism in C9orf72 repeat size is probably present, this study indicates that this is not likely to be of sufficient magnitude to result in a normal C9orf72 repeat length in blood but an abnormally expanded repeat length in the CNS. This suggests that a blood DNA test alone will usually be sufficient to make a diagnosis of C9orf72 repeat-related ALS. PMID:23894576

  15. Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

    PubMed Central

    Wang, Xin; Zhu, Nannan; Li, Wenwei; Zhu, Fanxiu; Wang, Yan; Yuan, Yan

    2015-01-01

    Herpesviruses acquire their envelope by budding into the lumen of cytoplasmic membrane vesicles. This process is initiated by component(s) on viral particles, which recognize the budding site where the viral glycoproteins are present and recruit cellular cargo transport and sorting machinery to the site to complete the budding process. Proteins in the tegument layer, connecting capsid and envelope, are candidates for the recognition of budding sites on vesicle membrane and induction of budding and final envelopment. We examined several outer and matrix tegument proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) and found that ORF45 associates with lipid rafts (LRs) of cellular membrane. LRs are membrane micro-domains, which have been implicated as relay stations in intracellular signaling and transport including viral entry and virion assembly. The ability of ORF45 to target LR is dependent on the mono-ubiquitylation of ORF45 at Lys297 as the mutation at Lys297 (K297R) abolished LR-association of ORF45. The K297R mutation also impairs ORF45 and viral particle co-localization with trans-Golgi network and endosomes, but facilitates ORF45 and viral particles co-localizing with lysosomes. More importantly, the recombinant KSHV carrying ORF45 K297R mutant (BAC-K297R) was found severely defective in producing mature and infectious virion particles in comparison to wild type KSHV (BAC16). Taken together, our results reveal a new function of KSHV tegument protein ORF45 in targeting LR of host cell membrane, promoting viral particles co-localization with trans-Golgi and endosome vesicles and facilitating the maturation and release of virion particles, suggesting that ORF45 plays a role in bringing KSHV particles to the budding site on cytoplasmic vesicle membrane and triggering the viral budding process for final envelopment and virion maturation. PMID:26650119

  16. Mycovirus-like DNA virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients.

    PubMed

    Lamberto, Iranzu; Gunst, Karin; Müller, Hermann; Zur Hausen, Harald; de Villiers, Ethel-Michele

    2014-01-01

    Myco-like viruses have been isolated from fungi, feces of various animals, and plant leaves. We report here the isolation of 3 complete genome sequences of gemycircularvirus-related viruses from healthy bovine serum and human brain and serum samples from patients with multiple sclerosis (MS). Their putative capsid proteins share similarity to Torque teno virus (TTV) open reading frame 1 (ORF1) proteins. PMID:25169858

  17. Mycovirus-Like DNA Virus Sequences from Cattle Serum and Human Brain and Serum Samples from Multiple Sclerosis Patients

    PubMed Central

    Lamberto, Iranzu; Gunst, Karin; Müller, Hermann; zur Hausen, Harald

    2014-01-01

    Myco-like viruses have been isolated from fungi, feces of various animals, and plant leaves. We report here the isolation of 3 complete genome sequences of gemycircularvirus-related viruses from healthy bovine serum and human brain and serum samples from patients with multiple sclerosis (MS). Their putative capsid proteins share similarity to Torque teno virus (TTV) open reading frame 1 (ORF1) proteins. PMID:25169858

  18. 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

  19. Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

    PubMed Central

    Broyles, S S; Moss, B

    1987-01-01

    Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites. Images PMID:2437324

  20. 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

  1. 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

  2. C9ORF72 hexanucleotide repeats in behavioral and motor neuron disease: clinical heterogeneity and pathological diversity

    PubMed Central

    Yokoyama, Jennifer S; Sirkis, Daniel W; Miller, Bruce L

    2014-01-01

    Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of frontotemporal dementia (FTD), a predominantly behavioral disease, and amyotrophic lateral sclerosis (ALS), a disease of motor neurons. The primary objectives of this review are to highlight the clinical heterogeneity associated with C9ORF72 pathogenic expansion and identify potential molecular mechanisms underlying selective vulnerability of distinct neural populations. The proposed mechanisms by which C9ORF72 expansion causes behavioral and motor neuron disease highlight the emerging role of impaired RNA and protein homeostasis in a spectrum of neurodegeneration and strengthen the biological connection between FTD and ALS. PMID:24753999

  3. Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system.

    PubMed

    Kobayashi, Tominari; Takahashi, Masaharu; Tanggis; Mulyanto; Jirintai, Suljid; Nagashima, Shigeo; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV. PMID:26992654

  4. Four novel C20orf54 mutations identified in Brown-Vialetto-Van Laere syndrome patients.

    PubMed

    Dezfouli, Mitra Ansari; Yadegari, Samira; Nafissi, Shahriar; Elahi, Elahe

    2012-09-01

    Brown-Vialetto-Van Laere syndrome (BVVLS) is a very rare neurodegenerative disorder characterized by pontobulbar palsy and sensorineural hearing loss. Its mode of inheritance in affected families has usually been autosomal recessive, although autosomal dominant inheritance and incomplete penetrance have also been reported. Recently, C20orf54 was identified as a causative gene for BVVLS. Twelve different mutations have so far been identified in 10 patients affected with BVVLS or the related disorder Fazio Londe syndrome. Here, results of screening of C20orf54 in three unrelated BVVLS patients are reported. Four novel mutations that affect amino acid changes, p.Asn21Ser, p.Pro220His, p.Ala312Val and p.Gly375Asp, were identified in the patients. The causative nucleotide variations were not observed in 200 control individuals. One of the patients harbored compound heterozygous mutations, but only one mutated allele was observed in each of the two remaining patients. PMID:22718020

  5. C19orf12 mutation leads to Karak pallido-pyramidal syndrome

    PubMed Central

    Kruer, Michael C.; Salih, Mustafa A.; Mooney, Catherine; Alzahrani, Jawahir; Elmalik, Salah A.; Kabiraj, Mohammad M.; Khan, Arif O.; Paudel, Reema; Houlden, Henry; Azzedine, Hamid; Alkuraya, Fowzan

    2015-01-01

    Pallido-pyramidal syndromes combine dystonia with or without parkinsonism and spasticity as part of a mixed neurodegenerative disorder. Several causative genes have been shown to lead to pallido-pyramidal syndromes, including FBXO7, ATP13A2, PLA2G6, PRKN and SPG11. In particular, mutations in PLA2G6 have been identified in patients with Karak syndrome, a neurodegenerative disorder that features ataxia, dystonia-parkinsonism, dementia and spasticity with neuroradiologic evidence of cerebellar atrophy and/or brain iron deposition. Some patients with phenotypic Karak syndrome do not have demonstrable mutations in PLA2G6. Using homozygosity mapping and direct sequencing in a multiplex consanguineous Saudi Arabian family with Karak syndrome, we identified a homozygous p.G53R mutation in C19orf12. Our findings expand the phenotypic spectrum associated with C19orf12 mutations. PMID:24361204

  6. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

    PubMed Central

    Ridler, Charlotte E.; Clayton, Emma L.; Devoy, Anny; Moens, Thomas; Norona, Frances E.; Woollacott, Ione O.C.; Pietrzyk, Julian; Cleverley, Karen; Nicoll, Andrew J.; Pickering-Brown, Stuart; Dols, Jacqueline; Cabecinha, Melissa; Hendrich, Oliver; Fratta, Pietro; Fisher, Elizabeth M.C.; Partridge, Linda; Isaacs, Adrian M.

    2016-01-01

    An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration. PMID:25103406

  7. Transcription Interference and ORF Nature Strongly Affect Promoter Strength in a Reconstituted Metabolic Pathway

    PubMed Central

    Carquet, Marie; Pompon, Denis; Truan, Gilles

    2015-01-01

    Fine tuning of individual enzyme expression level is necessary to alleviate metabolic imbalances in synthetic heterologous pathways. A known approach consists of choosing a suitable combination of promoters, based on their characterized strengths in model conditions. We questioned whether each step of a multiple-gene synthetic pathway could be independently tunable at the transcription level. Three open reading frames, coding for enzymes involved in a synthetic pathway, were combinatorially associated to different promoters on an episomal plasmid in Saccharomyces cerevisiae. We quantified the mRNA levels of the three genes in each strain of our generated combinatorial metabolic library. Our results evidenced that the ORF nature, position, and orientation induce strong discrepancies between the previously reported promoters’ strengths and the observed ones. We conclude that, in the context of metabolic reconstruction, the strength of usual promoters can be dramatically affected by many factors. Among them, transcriptional interference and ORF nature seem to be predominant. PMID:25767795

  8. 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

  9. Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

    PubMed Central

    Meulenberg, J J; Bende, R J; Pol, J M; Wensvoort, G; Moormann, R J

    1995-01-01

    The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV. PMID:8574824

  10. 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

  11. 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

  12. Emerging role of RNA•DNA hybrids in C9orf72-linked neurodegeneration.

    PubMed

    Wang, Jiou; Haeusler, Aaron R; Simko, Eric A J

    2015-01-01

    RNA plays an active role in structural polymorphism of the genome through the formation of stable RNA•DNA hybrids (R-loops). R-loops can modulate normal physiological processes and are also associated with pathological conditions, such as those related to nucleotide repeat expansions. A guanine-rich hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been linked to a spectrum of neurological conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we discuss the possible roles, both locally and genome-wide, of R-loops that may arise from the C9orf72 hexanucleotide repeat. R-loops have the potential to influence the pathological processes identified in many repeat expansion diseases, such as repeat instability, transcriptional dysregulation, epigenetic modification, and antisense-mediated gene regulation. We propose that, given the wide-ranging consequences of R-loops in the cell, these structures could underlie multiple pathological processes in C9orf72-linked neurodegeneration. PMID:25590632

  13. C9orf72 repeat expansions are restricted to the ALS-FTD spectrum.

    PubMed

    Ticozzi, Nicola; Tiloca, Cinzia; Calini, Daniela; Gagliardi, Stella; Altieri, Alessandra; Colombrita, Claudia; Cereda, Cristina; Ratti, Antonia; Pezzoli, Gianni; Borroni, Barbara; Goldwurm, Stefano; Padovani, Alessandro; Silani, Vincenzo

    2014-04-01

    Expansion of a GGGGCC repeat (RE) in the C9orf72 gene has been recently reported as the main genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the growing evidence of genetic and clinicopathologic overlap among ALS, FTD, and other neurodegenerative diseases, we investigated the occurrence of RE in a subset of 9 patients with ALS-plus syndromes, including Parkinson's disease (PD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and multiple system atrophy. We identified RE in 2 ALS-plus individuals (22.2%) displaying PSP and CBS features. On the basis of this finding, we extended our analysis to a cohort composed of 190 PD, 103 CBS, 107 PSP, and 177 Alzheimer's disease cases. We did not identify any RE in these patients, indicating that C9orf72 is in all probability not involved in the pathogenesis of these disorders. However, the high frequency of C9orf72 RE in patients with ALS-plus syndromes suggests that, similar to ALS-FTD patients, individuals with combined motor neuron and extrapyramidal features should be screened for RE, independent of their family history. PMID:24169076

  14. DENR•MCT-1 Promotes Translation Reinitiation Downstream of uORFs to Control Tissue Growth

    PubMed Central

    Schleich, Sibylle; Koledachkina, Tatyana; Miller, Katharine K.; Haneke, Katharina; Cheng, Yong-Sheng; Kuechler, Katrin; Stoecklin, Georg; Duncan, Kent E.; Teleman, Aurelio A.

    2014-01-01

    During cap-dependent eukaryotic translation initiation, ribosomes scan mRNA from the 5′ end to the first AUG start codon with favorable sequence context1,2. For many mRNAs this AUG belongs to a short upstream open reading frame (uORF)3, and translation of the main downstream ORF requires reinitiation, an incompletely understood process1,4-6. Reinitiation is thought to involve the same factors as standard initiation1,5,7. It is unknown if any factors specifically affect translation reinitiation without affecting standard cap-dependent translation. We uncover here the non-canonical initiation factors Density Regulated Protein (DENR) and Multiple Copies in T-cell Lymphoma-1 (MCT-1) as the first selective regulators of eukaryotic reinitiation. mRNAs containing upstream Open Reading Frames with strong Kozak sequences (stuORFs) selectively require DENR•MCT-1 for their proper translation, yielding a novel class of mRNAs that can be co-regulated and that is enriched for regulatory proteins such as oncogenic kinases. Collectively, our data reveal that cells have a previously unappreciated translational control system with a key role in supporting proliferation and tissue growth. PMID:25043021

  15. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases.

    PubMed

    Riboldi, Giulietta; Zanetta, Chiara; Ranieri, Michela; Nizzardo, Monica; Simone, Chiara; Magri, Francesca; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania

    2014-12-01

    Motor neuron disorders, and particularly amyotrophic lateral sclerosis (ALS), are fatal diseases that are due to the loss of motor neurons in the brain and spinal cord, with progressive paralysis and premature death. It has been recently shown that the most frequent genetic cause of ALS, frontotemporal dementia (FTD), and other neurological diseases is the expansion of a hexanucleotide repeat (GGGGCC) in the non-coding region of the C9ORF72 gene. The pathogenic mechanisms that produce cell death in the presence of this expansion are still unclear. One of the most likely hypotheses seems to be the gain-of-function that is achieved through the production of toxic RNA (able to sequester RNA-binding protein) and/or toxic proteins. In recent works, different authors have reported that antisense oligonucleotides complementary to the C9ORF72 RNA transcript sequence were able to significantly reduce RNA foci generated by the expanded RNA, in affected cells. Here, we summarize the recent findings that support the idea that the buildup of "toxic" RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS and also suggest that the use of antisense oligonucleotides targeting this transcript is a promising strategy for treating ALS/frontotemporal lobe dementia (FTLD) patients with the C9ORF72 repeat expansion. These data are particularly important, given the state of the art antisense technology, and they allow researchers to believe that a clinical application of these discoveries will be possible soon. PMID:24809691

  16. Precise limitation of concerted evolution to ORFs in mosquito Hsp82 genes.

    PubMed

    Benedict, M Q; Levine, B J; Ke, Z X; Cockburn, A F; Seawright, J A

    1996-02-01

    Two Hsp82 genes were isolated from the malaria vector Anopheles albimanus in a single lambda phage clone. The two genes are in a head-to-head arrangement separated by approx. 0.9 kbp. Northern hybridizations and 5' RACE demonstrate that both genes are transcribed, have moderate levels of constitutive transcription, and are also heat-inducible with maximum transcript accumulation occurring after 40 degrees C heat shocks. Both genes have typical heat-shock promoters and conserved intron boundaries in the untranslated leaders. The open reading frames are 99.6% identical differing in only nine silent nucleotide positions in the 2166 bp ORFs. However, precisely outside the ORFs, the flanking DNA of the two genes shows no evidence of common derivation. The high degree of identity between the two ORFs appears to be a result of gene conversion occurring by a process similar to that previously suspected in the A. albimanus Hsp70 genes and several D. melanogaster genes arranged as palindromes. This process probably involves a stem-loop intermediate and is restricted in extent by flanking sequence divergence. These Hsp82 genes clearly demonstrate the extreme precision with which gene conversion can lead to protein-coding-region homogeneity yet allow flanking DNA divergence. PMID:8630537

  17. Mutations in C12orf65 in Patients with Encephalomyopathy and a Mitochondrial Translation Defect

    PubMed Central

    Antonicka, Hana; Østergaard, Elsebet; Sasarman, Florin; Weraarpachai, Woranontee; Wibrand, Flemming; Pedersen, Anne Marie B.; Rodenburg, Richard J.; van der Knaap, Marjo S.; Smeitink, Jan A.M.; Chrzanowska-Lightowlers, Zofia M.; Shoubridge, Eric A.

    2010-01-01

    We investigated the genetic basis for a global and uniform decrease in mitochondrial translation in fibroblasts from patients in two unrelated pedigrees who developed Leigh syndrome, optic atrophy, and ophthalmoplegia. Analysis of the assembly of the oxidative phosphorylation complexes showed severe decreases of complexes I, IV, and V and a smaller decrease in complex III. The steady-state levels of mitochondrial mRNAs, tRNAs, and rRNAs were not reduced, nor were those of the mitochondrial translation elongation factors or the protein components of the mitochondrial ribosome. Using homozygosity mapping, we identified a 1 bp deletion in C12orf65 in one patient, and DNA sequence analysis showed a different 1 bp deletion in the second patient. Both mutations predict the same premature stop codon. C12orf65 belongs to a family of four mitochondrial class I peptide release factors, which also includes mtRF1a, mtRF1, and Ict1, all characterized by the presence of a GGQ motif at the active site. However, C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes. We suggest that it might play a role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation. PMID:20598281

  18. Mutation analysis of C9orf72 in patients with corticobasal syndrome.

    PubMed

    Anor, Cassandra J; Xi, Zhengrui; Zhang, Ming; Moreno, Danielle; Sato, Christine; Rogaeva, Ekaterina; Tartaglia, Maria Carmela

    2015-10-01

    Corticobasal syndrome (CBS) is a neurodegenerative disease characterized by progressive asymmetrical rigidity and apraxia, cortical sensory loss, myoclonus, dystonia, and cognitive impairment. CBS is usually sporadic and associated with tau pathology but there are reports of TDP-43 pathology. We screened 39 CBS cases to determine if any of the cases could be explained by a G4C2-repeat expansion in a noncoding region of C9orf72 gene, the most common genetic cause of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. One patient with CBS had a large (>50 repeats) expansion in C9orf72. Our case features a 63-year-old right-handed woman who developed mild apathy 9 years before presentation, which progressed to include behavioral symptoms, oral stereotypies, significant language impairment, parkinsonism and apraxia. A magnetic resonance imaging acquired at age 60 years, that is, 6 years after disease onset revealed significant asymmetric left > right frontotemporal atrophy, including orbitofrontal and parietal areas. Her father developed a behavioral syndrome and died at an early age. This case highlights the importance of genetic screening for C9orf72 in patients with CBS. PMID:26166205

  19. 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

  20. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease.

    PubMed

    Burberry, Aaron; Suzuki, Naoki; Wang, Jin-Yuan; Moccia, Rob; Mordes, Daniel A; Stewart, Morag H; Suzuki-Uematsu, Satomi; Ghosh, Sulagna; Singh, Ajay; Merkle, Florian T; Koszka, Kathryn; Li, Quan-Zhen; Zon, Leonard; Rossi, Derrick J; Trowbridge, Jennifer J; Notarangelo, Luigi D; Eggan, Kevin

    2016-07-13

    C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity. PMID:27412785

  1. Characterization of hepatitis E virus (HEV) from Algeria and Chad by partial genome sequence.

    PubMed

    van Cuyck-Gandré, H; Zhang, H Y; Tsarev, S A; Clements, N J; Cohen, S J; Caudill, J D; Buisson, Y; Coursaget, P; Warren, R L; Longer, C F

    1997-12-01

    The purpose of this study was to analyze partial nucleotide sequences and derived peptide sequences of hepatitis E virus (HEV) from two outbreaks of hepatitis E in Africa (Chad 1983-1984; Algeria 1978-1980). A portion of ORF3 and the major portion of ORF2 were amplified by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The PCR products were sequenced directly or after cloning into the pCRII vector. Sequences were then compared to the corresponding regions of reported full length HEV sequences. In the ORF2 and ORF3 regions, the homology between the Algerian and the Chad isolates at the nucleic acid level was 92 and 95%, respectively. At the peptide level the homology was 98% in both regions. In these regions, both strains are more related to Asian strains at the nucleic acid level (89 to 95%) and at the amino acid level (95 to 100%) than to the Mexico strain. At the peptide level the differences are less apparent. Both African isolates have amino acid changes in common with some reference strains although the Chad isolate has three unique changes. These African strains of HEV, based on the ORF2 and ORF3 phylogenetic trees, appear to be a distinct phylogenetic group, separate from the Mexican and Asian strains. PMID:9407381

  2. 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.

  3. MOLECULAR CHARACTERIZATION AND DETECTION OF PLUM BARK NECROSIS STEM PITTING-ASSOCIATED VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' abd 3' ends. PBNSPaV represents the si...

  4. Distribution, genetic diversity and recombination analysis of Citrus tristeza virus of India

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus tristeza virus (CTV) isolates representing all the citrus growing geographical zones of India were analyzed for sequence of the 5'ORF1a fragments of the partial LProI domain and for the coat protein (CP) gene. The sequences were compared with previously reported Indian and CTV genotypes from...

  5. Southern Tomato Virus: The Link between the Families Totiviridae and Partitiviridae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A dsRNA virus with a genome of 3.5 kb was isolated from field and greenhouse-grown tomato plants of different cultivars and geographic locations in North America. Cloning and sequencing of the viral genome showed the presence of two partially overlapping open reading frames (ORFs) and a genomic orga...

  6. 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...

  7. Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Lolium latent virus (LoLV, Lolavirus, Alphaflexiviridae) viral genome is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportion. The CP ORF contains two 5'-proximal AUGs, encoding Met 1 and Met 49, respectively promoting translation of th...

  8. Progressive amnestic dementia, hippocampal sclerosis, and mutation in C9ORF72.

    PubMed

    Murray, Melissa E; Bieniek, Kevin F; Banks Greenberg, M; DeJesus-Hernandez, Mariely; Rutherford, Nicola J; van Blitterswijk, Marka; Niemantsverdriet, Ellis; Ash, Peter E; Gendron, Tania F; Kouri, Naomi; Baker, Matt; Goodman, Ira J; Petrucelli, Leonard; Rademakers, Rosa; Dickson, Dennis W

    2013-10-01

    The most common cause of familial frontotemporal lobar degeneration with TAR DNA-binding protein-43 pathology (FTLD-TDP) has been found to be an expansion of a hexanucleotide repeat (GGGGCC) in a noncoding region of the gene C9ORF72. Hippocampal sclerosis (HpScl) is a common finding in FTLD-TDP. Our objective was to screen for the presence of C9ORF72 hexanucleotide repeat expansions in a pathologically confirmed cohort of "pure" hippocampal sclerosis cases (n = 33), outside the setting of FTLD-TDP and Alzheimer's disease (AD). Using a recently described repeat-associated non-ATG (RAN) translation (C9RANT) antibody that was found to be highly specific for c9FTD/ALS, we identified a single "pure" HpScl autopsy case with a repeat expansion in C9ORF72 (c9HpScl). Mutation screening was also performed with repeat-primed polymerase chain reaction and further confirmed with Southern blotting. The c9HpScl patient had a 14-year history of a slowly progressive amnestic syndrome and a clinical diagnosis of probable AD. Neuropsychological testing revealed memory impairment, but no deficits in other cognitive domains. Autopsy showed hippocampal sclerosis with TDP-43 immunoreactive neuronal inclusions relatively limited to limbic lobe structures. Neuritic pathology immunoreactive for p62 was more frequent than TDP-43 in amygdala and hippocampus. Frequent p62-positive neuronal inclusions were present in cerebellar granule neurons as is typical of C9ORF72 mutation carriers. There was no significant FTLD or motor neuron disease. C9RANT was found to be sensitive and specific in this autopsy-confirmed series of HpScl cases. The findings in this patient suggest that the clinical and pathologic spectrum of C9ORF72 repeat expansion is wider than frontotemporal dementia and motor neuron disease, including cases of progressive amnestic dementia with restricted TDP-43 pathology associated with HpScl. PMID:23922030

  9. 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

  10. 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

  11. Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea

    PubMed Central

    2010-01-01

    Background The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility. Results It was shown that the ORF220 protein can be guided to the mitochondria using the mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic stem mustard plants expressed the chimeric gene containing the orf220 gene and a mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic plants were male-sterile, most being unable to produce pollen while some could only produce non-vigorous pollen. The transgenic stem mustard plants also showed aberrant floral development identical to that observed in the CMS stem mustard phenotype. Results obtained from oligooarray analysis showed that some genes related to mitochondrial energy metabolism were down-regulated, indicating a weakening of mitochondrial function in transgenic stem mustard. Some genes related to pollen development were shown to be down-regulated in transgenic stem mustard and the expression of some transcription factor genes was also altered. Conclusion The work presented furthers our understanding of how the mitochondrially-targeted expression of CMS-associated orf220 gene causes male sterility through

  12. The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation.

    PubMed

    Sohn, Sook-Young; Hearing, Patrick

    2016-06-14

    The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses. PMID:27247387

  13. 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

  14. Culture systems for hepatitis E virus.

    PubMed

    Okamoto, Hiroaki

    2013-02-01

    The lack of an efficient cell culture system for hepatitis E virus (HEV) has greatly hampered detailed analyses of this virus. The first efficient cell culture systems for HEV that were developed were capable of secreting infectious HEV progenies in high titers into culture media, using PLC/PRF/5 cells derived from human hepatocellular carcinoma and A549 cells derived from human lung cancer as host cells. The success achieved with the original genotype 3 JE03-1760F strain has now been extended to various HEV strains in fecal and serum samples obtained from hepatitis E patients and to HEV strains in fecal and serum samples and liver tissues obtained from pigs and wild boar across species barriers. In addition, infectious HEV cDNA clones of the wild-type JE03-1760F strain and its variants have been engineered. Cell culture-generated HEV particles and those in circulating blood were found to be associated with lipids and open reading frame 3 (ORF3) protein, thereby likely contributing to the assembly and release of HEV from infected cells both in vivo and in vitro. The ORF3 protein interacts with the tumor susceptibility gene 101, a critical cellular protein required for the budding of enveloped viruses, through the Pro, Ser, Ala, and Pro (PSAP) motif in infected cells; ORF3 is co-localized with multivesicular bodies (MVBs) in the cytoplasm of infected cells, thus suggesting that HEV requires the MVB pathway for the egress of virus particles. This article reviews the development of efficient cell culture systems for a wide variety of infectious HEV strains obtained from humans, pigs, and wild boar, and also provides details of a new model for virion egress. PMID:23104469

  15. 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

  16. Constructional apraxia in frontotemporal dementia associated with the C9orf72 mutation: broadening the clinical and neuropsychological phenotype.

    PubMed

    Floris, Gianluca; Borghero, Giuseppe; Cannas, Antonino; Di Stefano, Francesca; Ruiu, Elisa; Murru, Maria R; Corongiu, Daniela; Cuccu, Stefania; Tranquilli, Stefania; Sardu, Claudia; Marrosu, Maria G; Chiò, Adriano; Marrosu, Francesco

    2015-03-01

    In our study we analysed clinical and neuropsychological data in a cohort of 57 Sardinian patients with FTD (55 apparently unrelated and two belonging to the same family), who underwent genetic screening for the C9orf72 mutation. Eight out of 56 patients were found positive for the C9orf72 mutation representing 14% of the entire cohort and 31.6% of the familial cases (6/19). C9orf72 mutated patients differed from the other FTD cases of the cohort for a younger age of onset, higher frequency of familial history for FTD and higher prevalence of delusional psychotic symptoms and hallucinations. In the neuropsychological assessment, C9orf72 mutated patients differed from non-mutated for the high frequency of visuospatial dysfunction regarding constructional apraxia (p = 0.02). In conclusion, our study confirms that Sardinian FTD patients have peculiar genetic characteristics and that C9orf72 mutated patients have a distinctive clinical and neuropsychological profile that could help differentiate them from other FTD patients. In our cohort we found that constructional apraxia, rarely reported in FTD, can properly discriminate between C9orf72 mutated and non-mutated patients and contribute to broaden the neuropsychological profile in frontotemporal dementia associated with this mutation. PMID:25285776

  17. MutS HOMOLOG1 silencing mediates ORF220 substoichiometric shifting and causes male sterility in Brassica juncea

    PubMed Central

    Zhao, Na; Xu, Xinyue; Wamboldt, Yashitola; Mackenzie, Sally A.; Hu, Zhongyuan; Yang, Jinghua; Zhang, Mingfang

    2016-01-01

    Cytoplasmic male sterility (CMS) has consistently been associated with the expression of mitochondrial open reading frames (ORFs) that arise from genomic rearrangements. Spontaneous fertility reversion in CMS has been observed in several cases, but a clear understanding of fertility reversion controlled by nuclear genetic influences has been lacking. Here, we identified spontaneous fertile revertant lines for Brassica juncea CMS cytoplasm in which the mitochondrial genome has undergone substoichiometric shifting (SSS) to suppress ORF220 copy number. We placed ORF220, with or without a mitochondrial targeting presequence, under the control of the CaMV35S and AP3 promoters in Arabidopsis to confirm that ORF220 causes male sterility when mitochondrially localized. We found that copy number of the ORF220 gene was altered under conditions that suppress MSH1, a nuclear gene that controls illegitimate recombination in plant mitochondria. MSH1-RNAi lines with increased ORF220 copy number were male sterile compared with wild type. We found that a wide range of genes involved in anther development were up- and down-regulated in revertant and MSH1-RNAi lines, respectively. The system that we have developed offers valuable future insight into the interplay of MSH1 and SSS in CMS induction and fertility reversion as a mediator of nuclear–mitochondrial crosstalk. PMID:26516127

  18. 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

  19. 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

  20. 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

  1. C2orf62 and TTC17 are involved in actin organization and ciliogenesis in zebrafish and human.

    PubMed

    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

  2. C11orf83, a mitochondrial cardiolipin-binding protein involved in bc1 complex assembly and supercomplex stabilization.

    PubMed

    Desmurs, Marjorie; Foti, Michelangelo; Raemy, Etienne; Vaz, Frédéric Maxime; Martinou, Jean-Claude; Bairoch, Amos; Lane, Lydie

    2015-04-01

    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

  3. 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

  4. 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. PMID:25701744

  5. Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate.

    PubMed

    Sudarshana, M R; Berger, P H

    1998-01-01

    The complete sequence of a North American tobacco rattle virus (TRV) isolate, 'Oregon yellow' (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 to TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than--SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses. ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses is also discussed. PMID:9739332

  6. Chimeric porcine reproductive and respiratory syndrome virus containing shuffled multiple envelope genes confers cross-protection in pigs.

    PubMed

    Tian, Debin; Ni, Yan-Yan; Zhou, Lei; Opriessnig, Tanja; Cao, Dianjun; Piñeyro, Pablo; Yugo, Danielle M; Overend, Christopher; Cao, Qian; Lynn Heffron, C; Halbur, Patrick G; Pearce, Douglas S; Calvert, Jay G; Meng, Xiang-Jin

    2015-11-01

    The extensive genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains is a major obstacle for vaccine development. We previously demonstrated that chimeric PRRSVs in which a single envelope gene (ORF3, ORF4, ORF5 or ORF6) was shuffled via DNA shuffling had an improved heterologous cross-neutralizing ability. In this study, we incorporate all of the individually-shuffled envelope genes together in different combinations into an infectious clone backbone of PRRSV MLV Fostera(®) PRRS. Five viable progeny chimeric viruses were rescued, and their growth characteristics were characterized in vitro. In a pilot pig study, two chimeric viruses (FV-SPDS-VR2,FV-SPDS-VR5) were found to induce cross-neutralizing antibodies against heterologous strains. A subsequent vaccination/challenge study in 72 pigs revealed that chimeric virus FV-SPDS-VR2 and parental virus conferred partial cross-protection when challenged with heterologous strains NADC20 or MN184B. The results have important implications for future development of an effective PRRSV vaccine that confers heterologous protection. PMID:26342466

  7. Open Reading Frame S/L of Varicella-Zoster Virus Encodes a Cytoplasmic Protein Expressed in Infected Cells

    PubMed Central

    Kemble, George W.; Annunziato, Paula; Lungu, Octavian; Winter, Ruth E.; Cha, Tai-An; Silverstein, Saul J.; Spaete, Richard R.

    2000-01-01

    We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3′ untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation. PMID:11070031

  8. Comprehensive annotation of Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach.

    PubMed

    Abd-Alla, Adly M M; Kariithi, Henry M; Cousserans, François; Parker, Nicolas J; İnce, İkbal Agah; Scully, Erin D; Boeren, Sjef; Geib, Scott M; Mekonnen, Solomon; Vlak, Just M; Parker, Andrew G; Vreysen, Marc J B; Bergoin, Max

    2016-04-01

    Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies. PMID:26801744

  9. Oligogenic inheritance of optineurin (OPTN) and C9ORF72 mutations in ALS highlights localisation of OPTN in the TDP-43-negative inclusions of C9ORF72-ALS.

    PubMed

    Bury, Joanna J; Highley, J Robin; Cooper-Knock, Johnathan; Goodall, Emily F; Higginbottom, Adrian; McDermott, Christopher J; Ince, Paul G; Shaw, Pamela J; Kirby, Janine

    2016-04-01

    Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss resulting in muscle weakness, spasticity and ultimately death. 5-10% are caused by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. Rarer genetic causes of ALS include mutation of optineurin (mt OPTN). Furthermore, optineurin protein has been localized to the ubiquitylated aggregates in several neurodegenerative diseases, including ALS. This study: (i) investigated the frequency of mt OPTN in ALS patients in England; (ii) characterized the clinical and neuropathological features of ALS associated with a mt OPTN; and (iii) investigated optineurin neuropathology in C9ORF72-related ALS (C9ORF72-ALS). We identified a heterozygous p.E322K missense mutation in exon 10 of OPTN in one familial ALS patient who additionally had a C9ORF72 mutation. This patient had bulbar, limb and respiratory disease without cognitive problems. Neuropathology revealed motor neurone loss, trans-activation response DNA protein 43 (TDP-43)-positive neuronal and glial cytoplasmic inclusions together with TDP-43-negative neuronal cytoplasmic inclusions in extra motor regions that are characteristic of C9ORF72-ALS. We have demonstrated that both TDP-43-positive and negative inclusion types had positive staining for optineurin by immunohistochemistry. We went on to show that optineurin was present in TDP-43-negative cytoplasmic extra motor inclusions in C9ORF72-ALS cases that do not carry mt OPTN. We conclude that: (i) OPTN mutations are associated with ALS; (ii) optineurin protein is present in a subset of the extramotor inclusions of C9ORF72-ALS; (iii) It is not uncommon for multiple ALS-causing mutations to occur in the same patient; and (iv) studies of optineurin are likely to provide useful dataregarding the pathophysiology of ALS and neurodegeneration. PMID:26303227

  10. Analysis of new aphid lethal paralysis virus (ALPV) isolates suggests evolution of two ALPV species.

    PubMed

    Liu, Sijun; Vijayendran, Diveena; Carrillo-Tripp, Jimena; Miller, W Allen; Bonning, Bryony C

    2014-12-01

    Aphid lethal paralysis virus (ALPV; family Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new isolate of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has an ssRNA genome of 9940 nt. Based on phylogenetic analysis, ALPV-AP was closely related to ALPV-AM, an ALPV isolate from honeybees, Apis mellifera, in Spain and Brookings, SD, USA. The distinct evolutionary branches suggested the existence of two lineages of the ALPV virus. One consisted of ALPV-AP and ALPV-AM, whilst all other isolates of ALPV grouped into the other lineage. The similarity of ALPV-AP and ALPV-AM was up to 88 % at the RNA level, compared with 78-79 % between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM was 98-99 % for both ORF1 and ORF2, whilst only 85-87 % for ORF1 and 91-92 % for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE (rapid amplification of cDNA ends) products and cDNA clones of the virus genome revealed sequence variation in the 5' UTRs and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicated that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and we propose that ALPV isolates be classified as two separate viral species. PMID:25170050

  11. The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway.

    PubMed

    Sullivan, Peter M; Zhou, Xiaolai; Robins, Adam M; Paushter, Daniel H; Kim, Dongsung; Smolka, Marcus B; Hu, Fenghua

    2016-01-01

    Hexanucleotide repeat expansion in the C9orf72 gene is a leading cause of frontotemporal lobar degeneration (FTLD) with amyotrophic lateral sclerosis (ALS). Reduced expression of C9orf72 has been proposed as a possible disease mechanism. However, the cellular function of C9orf72 remains to be characterized. Here we report the identification of two binding partners of C9orf72: SMCR8 and WDR41. We show that WDR41 interacts with the C9orf72/SMCR8 heterodimer and WDR41 is tightly associated with the Golgi complex. We further demonstrate that C9orf72/SMCR8/WDR41 associates with the FIP200/Ulk1 complex, which is essential for autophagy initiation. C9orf72 deficient mice, generated using the CRISPR/Cas9 system, show severe inflammation in multiple organs, including lymph node, spleen and liver. Lymph node enlargement and severe splenomegaly are accompanied with macrophage infiltration. Increased levels of autophagy and lysosomal proteins and autophagy defects were detected in both the spleen and liver of C9orf72 deficient mice, supporting an in vivo role of C9orf72 in regulating the autophagy/lysosome pathway. In summary, our study elucidates potential physiological functions of C9orf72 and disease mechanisms of ALS/FTLD. PMID:27193190

  12. 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

  13. 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

  14. 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

  15. Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms

    PubMed Central

    Verweij, Marieke C.; Wellish, Mary; Whitmer, Travis; Malouli, Daniel; Lapel, Martin; Jonjić, Stipan; Haas, Juergen G.; DeFilippis, Victor R.; Mahalingam, Ravi; Früh, Klaus

    2015-01-01

    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. PMID:25973608

  16. 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

  17. Delineation of C12orf65-related phenotypes: a genotype–phenotype relationship

    PubMed Central

    Spiegel, Ronen; Mandel, Hanna; Saada, Ann; Lerer, Issy; Burger, Ayala; Shaag, Avraham; Shalev, Stavit A; Jabaly-Habib, Haneen; Goldsher, Dorit; Gomori, John M; Lossos, Alex; Elpeleg, Orly; Meiner, Vardiella

    2014-01-01

    C12orf65 participates in the process of mitochondrial translation and has been shown to be associated with a spectrum of phenotypes, including early onset optic atrophy, progressive encephalomyopathy, peripheral neuropathy, and spastic paraparesis.We used whole-genome homozygosity mapping as well as exome sequencing and targeted gene sequencing to identify novel C12orf65 disease-causing mutations in seven affected individuals originating from two consanguineous families. In four family members affected with childhood-onset optic atrophy accompanied by slowly progressive peripheral neuropathy and spastic paraparesis, we identified a homozygous frame shift mutation c.413_417 delAACAA, which predicts a truncated protein lacking the C-terminal portion. In the second family, we studied three affected individuals who presented with early onset optic atrophy, peripheral neuropathy, and spastic gait in addition to moderate intellectual disability. Muscle biopsy in two of the patients revealed decreased activities of the mitochondrial respiratory chain complexes I and IV. In these patients, we identified a homozygous splice mutation, g.21043 T>A (c.282+2 T>A) which leads to skipping of exon 2. Our study broadens the phenotypic spectrum of C12orf65 defects and highlights the triad of optic atrophy, axonal neuropathy and spastic paraparesis as its key clinical features. In addition, a clear genotype–phenotype correlation is anticipated in which deleterious mutations which disrupt the GGQ-containing domain in the first coding exon are expected to result in a more severe phenotype, whereas down-stream C-terminal mutations may result in a more favorable phenotype, typically lacking cognitive impairment. PMID:24424123

  18. The C9ORF72 expansion mutation: gene structure, phenotypic and diagnostic issues.

    PubMed

    Woollacott, Ione O C; Mead, Simon

    2014-03-01

    The discovery of the C9ORF72 hexanucleotide repeat expansion in 2011 and the immediate realisation of a remarkably high prevalence in both familial and sporadic frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) triggered an explosion of interest in studies aiming to define the associated clinical and investigation phenotypes and attempts to develop technologies to measure more accurately the size of the repeat region. This article reviews progress in these areas over the subsequent 2 years, focussing on issues directly relevant to the practising physician. First, we summarise findings from studies regarding the global prevalence of the expansion, not only in FTLD and ALS cases, but also in other neurological diseases and its concurrence with other genetic mutations associated with FTLD and ALS. Second, we discuss the variability in normal repeat number in cases and controls and the theories regarding the relevance of intermediate and pathological repeat number for disease risk and clinical phenotype. Third, we discuss the usefulness of various features within the FTLD and ALS clinical phenotype in aiding differentiation between cases with and without the C9ORF72 expansion. Fourth, we review clinical investigations used to identify cases with the expansion, including neuroimaging and cerebrospinal fluid markers, and describe the mechanisms and limitations of the various diagnostic laboratory techniques used to quantify repeat number in cases and controls. Finally, we discuss the issues surrounding accurate clinical and technological diagnosis of patients with FTLD and/or ALS associated with the C9ORF72 expansion, and outline areas for future research that might aid better diagnosis and genetic counselling of patients with seemingly sporadic or familial FTLD or ALS and their relatives. PMID:24515836

  19. Jump from Pre-mutation to Pathologic Expansion in C9orf72

    PubMed Central

    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

    2015-01-01

    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

  20. 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

  1. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein.

    PubMed

    Rahaman, Siti Nurulnabila A; Mat Yusop, Jastina; Mohamed-Hussein, Zeti-Azura; Ho, Kok Lian; Teh, Aik-Hong; Waterman, Jitka; Ng, Chyan Leong

    2016-03-01

    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%. PMID:26919524

  2. Erratum: C9ORF72 expression and cellular localization over mouse development.

    PubMed

    Atkinson, Rachel A K; Fernandez-Martos, Carmen M; Atkin, Julie D; Vickers, James C; King, Anna E

    2016-01-01

    After publication of this article it was noticed there was an error in the Methods section under the subsection: Protein extraction and western blot analysis. The text including the error is as follows: "Denatured protein samples (15 μg) from each time-point were electrophoresed into 10 % SDS-PAGE gels (BioRad), transferred to PVDF membranes (BioRad) and incubated in primary antibodies overnight (Table 1)". Instead it should read: "…antibodies, C9ORF72 (1:500, Santa Cruz, sc-138763) and GAPDH (1:7000, Millipore), overnight." This error has since been updated in the article. PMID:26727886

  3. Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus

    PubMed Central

    Nair, Vidya P.; Madhvi, Abhilasha; Bakshi, Karishma; Srivastava, Akriti; Shalimar; Nayak, Baibaswata; CT, Ranjith Kumar; Surjit, Milan

    2016-01-01

    Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the

  4. Autogenous Translational Regulation of the Borna Disease Virus Negative Control Factor X from Polycistronic mRNA Using Host RNA Helicases

    PubMed Central

    Watanabe, Yohei; Ohtaki, Naohiro; Hayashi, Yohei; Ikuta, Kazuyoshi; Tomonaga, Keizo

    2009-01-01

    Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5′ to 3′ direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5′ untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases. PMID:19893625

  5. 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

  6. 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

  7. Early dipeptide repeat pathology in a frontotemporal dementia kindred with C9ORF72 mutation and intellectual disability.

    PubMed

    Proudfoot, Malcolm; Gutowski, Nick J; Edbauer, Dieter; Hilton, David A; Stephens, Mark; Rankin, Julia; Mackenzie, Ian R A

    2014-03-01

    Familial cases of frontotemporal dementia (FTD) provide an opportunity to study the pathophysiology of this clinically diverse condition. The C9ORF72 mutation is the most common cause of familial FTD, recent pathological descriptions challenge existing TDP-43 based hypotheses of sporadic FTD pathogenesis. Non-ATG dependent translation of the hexanucleotide expansion into aggregating dipeptide repeat (DPR) proteins may represent a novel pathomechanism. We report detection of the DPR aggregates very early in C9ORF72 FTD development and also describe childhood intellectual disability as a clinical feature preceding dementia. The index case presented with psychiatric symptoms, progressing into typical FTD. Autopsy revealed extensive neuronal DPR aggregates but only minimal TDP-43 pathology. Her intellectually disabled elder son, also carrying the C9ORF72 mutation, died aged 26 years and at autopsy only DPR aggregates without TDP-43 were found. A second son also has intellectual disability, his C9ORF72 status is unknown, but chromosomal microarray revealed no other cause of disability. These cases both extend the existing phenotype of C9ORF72 mutation and highlight the potential significance of DPR translation early in disease development. PMID:24445903

  8. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair.

    PubMed

    Craxton, A; Somers, J; Munnur, D; Jukes-Jones, R; Cain, K; Malewicz, M

    2015-06-01

    Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs-c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142-XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells. PMID:25941166

  9. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

    PubMed Central

    Craxton, A; Somers, J; Munnur, D; Jukes-Jones, R; Cain, K; Malewicz, M

    2015-01-01

    Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells. PMID:25941166

  10. Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition

    PubMed Central

    Goodier, John L.; Cheung, Ling E.; Kazazian, Haig H.

    2013-01-01

    LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic ‘parasites’. PMID:23749060

  11. 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

  12. C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

    PubMed

    Haeusler, Aaron R; Donnelly, Christopher J; Periz, Goran; Simko, Eric A J; Shaw, Patrick G; Kim, Min-Sik; Maragakis, Nicholas J; Troncoso, Juan C; Pandey, Akhilesh; Sattler, Rita; Rothstein, Jeffrey D; Wang, Jiou

    2014-03-13

    A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases. PMID:24598541

  13. 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

  14. 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

  15. C5orf42 is the major gene responsible for OFD syndrome type VI.

    PubMed

    Lopez, Estelle; Thauvin-Robinet, Christel; Reversade, Bruno; Khartoufi, Nadia El; Devisme, Louise; Holder, Muriel; Ansart-Franquet, Hélène; Avila, Magali; Lacombe, Didier; Kleinfinger, Pascale; Kaori, Irahara; Takanashi, Jun-Ichi; Le Merrer, Martine; Martinovic, Jelena; Noël, Catherine; Shboul, Mohammad; Ho, Lena; Güven, Yeliz; Razavi, Ferechté; Burglen, Lydie; Gigot, Nadège; Darmency-Stamboul, Véronique; Thevenon, Julien; Aral, Bernard; Kayserili, Hülya; Huet, Frédéric; Lyonnet, Stanislas; Le Caignec, Cédric; Franco, Brunella; Rivière, Jean-Baptiste; Faivre, Laurence; Attié-Bitach, Tania

    2014-03-01

    Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the "Joubert syndrome related disorders". Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies. PMID:24178751

  16. The Effect of Reading Duration on the Reliability and Validity of Middle School Students’ ORF Performance

    PubMed Central

    Barth, Amy E.; Stuebing, Karla K.; Fletcher, Jack M.; Denton, Carolyn A.; Vaughn, Sharon; Francis, David

    2014-01-01

    We evaluated the technical adequacy of oral reading fluency (ORF) probes in which 1,472 middle school students with and without reading difficulties read fluency probes for 60 s versus reading the full passage. Results suggested that the reliability of 60-s probes (rs ≥ .75) was not substantively different than full passage probes (rs ≥ .77) among struggling readers and typically developing readers in Grades 6 to 8. The correlation of 60-s and the full passage probes with norm-referenced measures of ORF ranged from .32 to .83, and the correlation with norm-referenced measures of reading comprehension ranged from .32 to .54, indicating that both measures were moderately valid and adequate for use among middle school students. Last, full passage probes with sensitivity rates ranging from .40 to .45 were only slightly more sensitive for identifying at-risk readers than 60-s probes, with sensitivity rates ranging from .36 to .40, suggesting that the full passage probes identified a slightly higher percentage of at-risk students with reading difficulties. PMID:25541580

  17. Distinct neurological disorders with C9orf72 mutations: genetics, pathogenesis, and therapy.

    PubMed

    Chi, Song; Jiang, Teng; Tan, Lan; Yu, Jin-Tai

    2016-07-01

    The G4C2 repeat expansion within C9orf72 has been recently identified as the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. This mutation has also been detected in a variety of other neurological diseases with distinct clinical manifestations. The exact mechanisms of how this mutation leads to the wide spectrum of clinical syndromes remain unknown. A series of molecular changes together with some potential modifiers may play a key role. Nucleolar stress, nucleocytoplasmic transport defect, oxidative damage, inhibited stress granules assembly, activated endoplasmic reticulum stress, and inhibited proteasome activity are mechanisms that contribute to the pathogenesis of these diseases. Additional mutations, epigenetic modifiers, and repeat size are potential modifiers that modulate specific phenotypes on the basis of the molecular changes. Here, we summarize distinct C9orf72-related neurological disorders and their corresponding neuropathological changes. Then, we elucidate the existing molecular knowledge and the potential modifiers. Finally, we detail the main target of treatment aiming at controlling expanded RNA transcripts. PMID:27139021

  18. 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

  19. 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. PMID:24496499

  20. Identification and genomic analysis of two duck-origin Tembusu virus strains in southern China.

    PubMed

    Li, Linlin; An, Hejia; Sun, Minhua; Dong, Jiawen; Yuan, Jianfeng; Hu, Qilin

    2012-08-01

    Egg-laying duck flocks in Guangdong province, southern China, have been suffering a widely spreading infectious disease with abrupt egg drops and death since the winter of 2010. However, the causative pathogen was not known. We obtained two independent virus isolates named FS and JM from the diseased layer duck flocks and identified them as duck-origin Tembusu virus by PCR detection, sequencing the entire length of the open reading frames (ORFs). The two isolates FS and JM shared high sequence similarity to the isolates of duck-origin Tembusu virus that was first emerged in eastern China in April 2010. Blast analysis shows that the whole ORF sequences of FS and JM have the highest similarity (>99 %) to BYD-1(the first reported duck-origin Tembusu virus) and JS804 (the first reported goose-origin Tembusu virus), indicating that the full-length genomes were highly conserved in waterfowl-origin Tembusu viruses. The present study suggests that duck-origin Tembusu viruses have spread fast from eastern China to southern China, causing widely spreading infections. The high conservation of duck-origin Tembusu virus strains provides the genomic basis for choosing the strains for vaccine preparation for better protection against this new virus infection. PMID:22581444

  1. Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

    PubMed Central

    Tang, Weiliang; Lázaro, Catherine A.; Campbell, Jean S.; Parks, W. Tony; Katze, Michael G.; Fausto, Nelson

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor κB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation. PMID:17991716

  2. Complete Genome Sequence of an NADC30-Like Porcine Reproductive and Respiratory Syndrome Virus Characterized by Recombination with Other Strains.

    PubMed

    Li, Yingying; Ji, Guobiao; Wang, Juan; Tan, Feifei; Zhuang, Jinshan; Li, Xiangdong; Tian, Kegong

    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

  3. 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

  4. 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

  5. An eriophyid mite-transmitted plant virus contains eight genomic RNA segments with unusual heterogeneity in the nucleocapsid protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eriophyid mite-transmitted, multipartite, negative-sense plant RNA viruses with membrane-bound spherical virions are classified in the genus Emaravirus. The relatively dissimilar emaraviruses evolved with 4 to 6 genomic RNAs with one open reading frame (ORF) in each genomic RNA segment. We report he...

  6. 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

  7. 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

  8. 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

  9. Complete genome sequence of keunjorong mosaic virus, a potyvirus from Cynanchum wilfordii.

    PubMed

    Nam, Moon; Lee, Joo-Hee; Choi, Hong Soo; Lim, Hyoun-Sub; Moon, Jae Sun; Lee, Su-Heon

    2013-08-01

    We have determined the complete genome sequence of keunjorong mosaic virus (KjMV). The KjMV genome is composed of 9,611 nucleotides, excluding the 3'-terminal poly(A) tail. It contains two open reading frames (ORFs), with the large one encoding a polyprotein of 3,070 amino acids and the small overlapping ORF encoding a PIPO protein of 81 amino acids. The KjMV genome shared the highest nucleotide sequence identity (57.5  %) with pepper mottle virus and freesia mosaic virus, two members of the genus Potyvirus. Based on the phylogenetic relatedness to known potyviruses, KjMV appears to be a member of a new species in the genus Potyvirus. PMID:23504105

  10. The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

    PubMed Central

    Visalli, Melissa A.; House, Brittany L.; Selariu, Anca; Zhu, Hua

    2014-01-01

    ABSTRACT The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. IMPORTANCE Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets

  11. Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2

    PubMed Central

    2010-01-01

    Background The capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in E. coli, for diagnosis of PCV infection. Results The ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%. Conclusions This ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera. PMID:20958981

  12. Amyotrophic Lateral Sclerosis with Frontotemporal Dementia in the Presence of C9orf72 Repeat Expansion—A Case Report

    PubMed Central

    Bonda, Chaitanya; Kolikonda, Murali K.; Brown, Martin E.

    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.

  13. ECHO virus

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that lead to ...

  14. 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. PMID:26092422

  15. Characterization of a Previously Unidentified Viral Protein in Porcine Circovirus Type 2-Infected Cells and Its Role in Virus-Induced Apoptosis

    PubMed Central

    Liu, Jue; Chen, Isabelle; Kwang, Jimmy

    2005-01-01

    Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways. PMID:15956572

  16. 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.

  17. Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae.

    PubMed

    Hajeri, Subhas; Ramadugu, Chandrika; Keremane, Manjunath; Vidalakis, Georgios; Lee, Richard

    2010-09-01

    The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3' poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5' and 541 nt at 3' termini. ORF1 potentially encoding a 227.48-kDa polyprotein, which has methyltransferase, oxygenase, endopeptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25 kDa, while ORF3 encodes a coat protein of 40.69 kDa. Protein database searches showed 98-99% matches of DMV ORFs with citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis based on the RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. DMV did not satisfy the molecular criteria for demarcation of an independent species within the genus Citrivirus, family Betaflexiviridae, and hence, DMV can be considered a CLBV isolate. PMID:20644968

  18. 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

  19. Frontotemporal lobar degeneration with TDP-43 proteinopathy and chromosome 9p repeat expansion in C9ORF72: clinicopathologic correlation.

    PubMed

    Bigio, Eileen H; Weintraub, Sandra; Rademakers, Rosa; Baker, Matt; Ahmadian, Saman S; Rademaker, Alfred; Weitner, Bing Bing; Mao, Qinwen; Lee, Kyung-Hwa; Mishra, Manjari; Ganti, Rakhee A; Mesulam, M-Marsel

    2013-04-01

    Mutations in C9ORF72 resulting in expanded hexanucleotide repeats were recently reported to be the underlying genetic abnormality in chromosome 9p-linked frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kD (TDP-43) proteinopathy (FTLD-TDP), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND). Several subsequent publications described the neuropathology as being similar to that of FTLD-TDP and ALS without C9ORF72 mutations, except that cases with mutations have p62 and ubiquitin positive, TDP-43 negative inclusions in cerebellum, hippocampus, neocortex, and basal ganglia. The identity of this protein is as yet unknown, and its significance is unclear. With the goal of potentially uncovering the significance of these inclusions, we compared the clinical, pathologic and genetic characteristics in cases with C9ORF72 mutations to those without. We confirmed the apparent specificity of p62 positive, TDP-43 negative inclusions to cases with C9ORF72 mutations. In hippocampus, these inclusions correlated with hippocampal atrophy. No additional correlations were uncovered. However, this is the first report to show that although most cases with C9ORF72 mutations were TDP type B, some of the pathologic characteristics in these cases were more similar to TDP types A and C than to type B cases. These include greater cortical and hippocampal atrophy, greater ventricular dilatation, more neuronal loss and gliosis in temporal lobe and striatum, and TDP-43 positive fine neuritic profiles in the hippocampus, implying that the C9ORF72 mutation modifies the pathologic phenotype of FTLD-TDP type B. PMID:22702520

  20. Rapid and simple detection of methicillin-resistance staphylococcus aureus by orfX loop-mediated isothermal amplification assay

    PubMed Central

    2014-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA). Results The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively. Conclusions The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA. PMID:24456841

  1. Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence.

    PubMed

    Wang, Yue; Liang, Yajie; Han, Jun; Burkhart, Kelly M; Vaughn, Eric M; Roof, Michael B; Faaberg, Kay S

    2008-02-20

    Two genetically distinct infectious recombinant virus clones (pMLV, constructed from Ingelvac PRRS MLV and pMN184, constructed from virulent strain MN184) were developed to study attenuation of contemporary porcine reproductive and respiratory syndrome virus (PRRSV) strain MN184. Two reciprocal chimeric clones (pMLVORF1/MN184 and pMN184ORF1/MLV) were then constructed, such that the 5'UTR/ORF1 of one genotype was linked to ORF2-7/3'UTR from the other genotype. In vitro studies demonstrated that the rescued chimeric viruses possessed intermediate growth properties compared to recombinant rMLV and rMN184. Swine inoculation with rMN184 and rMLV verified that these viruses fully mimicked the respective parent virus. In addition, earlier and higher antibody responses were detected in animals infected with rMN184 in contrast to those infected with rMLV. Chimeric virus treatment groups showed similar antibody responses as seen with these parent viruses, but much less severe pathogenesis when compared to the rMN184 group. These data suggested that genetic aspects of Ingelvac PRRS MLV 5'UTR/ORF1 replicase region and/or the structural proteins/3'UTR can serve to attenuate virulent strain MN184. The data also indicated that designed PRRSV vaccines could be developed, keeping the known 5'UTR/replicase region of an early vaccine strain such as Ingelvac PRRS MLV intact, but replacing the structural protein/3'UTR domain with that of an emerging virulent virus. PMID:17976680

  2. Subversion and piracy: DNA viruses and immune evasion.

    PubMed

    Haig, D M

    2001-06-01

    During the co-evolution of viruses with their vertebrate hosts, the DNA viruses have acquired an impressive array of immunomodulatory genes to combat host immune responses and their hosts have developed a sophisticated immune system to contain virus infections. In order to replicate, the viruses have evolved mechanisms to inhibit key host anti-virus responses that include apoptosis, interferon production, chemokine production, inflammatory cytokine production, and the activity of cytotoxic T-cells, natural killer cells and antibody. In addition, some of the viruses encode cytokine or chemokine homologues that recruit or expand cell numbers for infection or that subvert the host cellular response from a protective response to a benign one. The specificity of the viral immunomodulatory molecules reflects the life cycle and the pathogenesis of the viruses. Herpesviruses achieve latency in host cells by inducing cell survival and protecting infected cells from immune recognition. This involves interference with cell signal transduction pathways. Many of the viral immunomodulatory proteins are homologues of host proteins that appear to have been pirated from the host and reassorted in the virus genomes. Some of these have unique functions and indicate novel or important aspects of both viral pathogenesis and host immunity to viruses. The specific example of orf virus infection of sheep is described. PMID:11676616

  3. Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

    PubMed

    Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

    2015-08-01

    In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy. PMID:25872740

  4. 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. PMID:27236849

  5. The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Binds to HIF-1α and Causes Its Degradation via the Proteasome Pathway

    PubMed Central

    Kousoulas, Konstantin G.

    2013-01-01

    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

  6. 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

  7. Molecular characterisation of the full-length genome of olive latent virus 1 isolated from tomato.

    PubMed

    Hasiów-Jaroszewska, Beata; Borodynko, Natasza; Pospieszny, Henryk

    2011-05-01

    Olive latent virus 1 (OLV-1) is a species of the Necrovirus genus. So far, it has been reported to infect olive, citrus tree and tulip. Here, we determined and analysed the complete genomic sequence of an isolate designated as CM1, which was collected from tomato plant in the Wielkopolska region of Poland and represents the prevalent isolate of OLV-1. The CM1 genome consists of monopartite single-stranded positive-sense RNA genome sized 3,699 nt with five open reading frames (ORFs) and small inter-cistronic regions. ORF1 encodes a polypeptide with a molecular weight of 23 kDa and the read-through (RT) of its amber stop codon results in ORF1 RT that encodes the virus RNA-dependent RNA polymerase. ORF2 and ORF3 encode two peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in cell-to-cell movement. ORF4 is located in the 3' terminal and encodes a protein with 30 kDa identified as the viral coat protein (CP). The differences in CP region of four OLV-1 isolates whose sequences have been deposited in GenBank were observed. Nucleotide sequence identities of the CP of tomato CM1 isolate with those of olive, citrus and tulip isolates were 91.8%, 89.5% and 92.5%, respectively. In contrast to other OLV-1 isolates, CM1 induced necrotic spots on tomato plants and elicited necrotic local lesions on Nicotiana benthamiana, followed by systemic infection. This is the third complete genomic sequence of OLV-1 reported and the first one from tomato. PMID:21181333

  8. Attempts to enhance cross-protection against porcine reproductive and respiratory syndrome viruses using chimeric viruses containing structural genes from two antigenically distinct strains.

    PubMed

    Sun, Dong; Khatun, Amina; Kim, Won-Il; Cooper, Vickie; Cho, Yong-Il; Wang, Chong; Choi, Eun-Jin; Yoon, Kyoung-Jin

    2016-08-01

    Due to significant antigenic variations between field isolates of porcine reproductive and respiratory syndrome virus (PRRSV), suboptimal cross-protection between different viruses impedes the effective control of PRRS via vaccination. Our previous study showed that chimeric viruses containing mixed structural genes from two distinct strains (VR2332 and JA142) of PRRSV were highly susceptible to the viral neutralizing activity of antisera generated against both parental strains. In this study, three chimeric viruses (JAP5, JAP56 and JAP2-6) were constructed by replacing ORF5, ORFs 5 and 6, and ORFs 2-6 of VR2332 with the corresponding genes of JA142, respectively, and their ability to confer cross-protection against challenge with the VR2332 and JA142 strains was evaluated in vivo. A total of 114 pigs were divided into 6 groups, and each group was intramuscularly injected with one of the 3 chimeric viruses (n=16 pigs per group), VR2332 (n=24), JA142 (n=24), or sham inoculum (n=18). At 44days post-inoculation (dpi), these pigs were further divided into 15 groups (n=6 or 8 pigs per group) and intranasally challenged with VR2332, JA142, or sham inoculum. All pigs inoculated with one of the chimeric viruses prior to challenge had lower viremia levels than the challenge control pigs. Prior inoculation with JAP56 markedly decreased viremia to nearly undetectable levels in pigs challenged with either VR2332 or JA142. These results suggest that chimeric viruses harboring mixed structural genes from two distinct PRRSV strains can provide protection against both donor viruses. PMID:27406935

  9. Novel ssDNA viruses discovered in yellow-crowned parakeet (Cya