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Sample records for env gene proteins

  1. Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

    1991-01-01

    F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

  2. Identification and characterization of the Escherichia coli envC gene encoding a periplasmic coiled-coil protein with putative peptidase activity.

    PubMed

    Hara, Hiroshi; Narita, Setsuko; Karibian, Doris; Park, James T; Yamamoto, Yoshihiro; Nishimura, Yukinobu

    2002-07-02

    PM61 is a chain-forming envC strain of Escherichia coli with a leaky outer membrane. It was found to have an oversized penicillin-binding protein 3, which was the result of an IS4 insertion in the prc gene. The other properties of PM61 were caused by the envC mutation. We cloned the envC (yibP) gene and identified the mutation site, causing a single residue substitution, H366Y, in the PM61 envC allele. The gene product was predicted to be a periplasmic protein having coiled-coil structure in the N-terminal region and homology to lysostaphin in the C-terminal region. Overexpression of envC inhibited cell growth, and overexpression of the PM61 mutant allele caused cell lysis. Disruption of the chromosomal envC caused the same defects as the envC point mutation, indicating the gene is dispensable for growth but important for normal septation/separation and cell envelope integrity.

  3. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    SciTech Connect

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  4. Nucleotide Sequence of the Akv env Gene

    PubMed Central

    Lenz, Jack; Crowther, Robert; Straceski, Anthony; Haseltine, William

    1982-01-01

    The sequence of 2,191 nucleotides encoding the env gene of murine retrovirus Akv was determined by using a molecular clone of the Akv provirus. Deduction of the encoded amino acid sequence showed that a single open reading frame encodes a 638-amino acid precursor to gp70 and p15E. In addition, there is a typical leader sequence preceding the amino terminus of gp70. The locations of potential glycosylation sites and other structural features indicate that the entire gp70 molecule and most of p15E are located on the outer side of the membrane. Internal cleavage of the env precursor to generate gp70 and p15E occurs immediately adjacent to several basic amino acids at the carboxyl terminus of gp70. This cleavage generates a region of 42 uncharged, relatively hydrophobic amino acids at the amino terminus of p15E, which is located in a position analogous to the hydrophobic membrane fusion sequence of influenza virus hemagglutinin. The mature polypeptides are predicted to associate with the membrane via a region of 30 uncharged, mostly hydrophobic amino acids located near the carboxyl terminus of p15E. Distal to this membrane association region is a sequence of 35 amino acids at the carboxyl terminus of the env precursor, which is predicted to be located on the inner side of the membrane. By analogy to Moloney murine leukemia virus, a proteolytic cleavage in this region removes the terminal 19 amino acids, thus generating the carboxyl terminus of p15E. This leaves 15 amino acids at the carboxyl terminus of p15E on the inner side of the membrane in a position to interact with virion cores during budding. The precise location and order of the large RNase T1-resistant oligonucleotides in the env region were determined and compared with those from several leukemogenic viruses of AKR origin. This permitted a determination of how the differences in the leukemogenic viruses affect the primary structure of the env gene products. PMID:6283170

  5. Novel Feline Leukemia Virus Interference Group Based on the env Gene

    PubMed Central

    Miyake, Ariko; Watanabe, Shinya; Hiratsuka, Takahiro; Ito, Jumpei; Ngo, Minh Ha; Makundi, Isaac; Kawasaki, Junna; Endo, Yasuyuki; Tsujimoto, Hajime

    2016-01-01

    Feline leukemia virus (FeLV) subgroups have emerged in infected cats via the mutation or recombination of the env gene of subgroup A FeLV (FeLV-A), the primary virus. We report the isolation and characterization of a novel env gene, TG35-2, and report that the TG35-2 pseudotype can be categorized as a novel FeLV subgroup. The TG35-2 envelope protein displays strong sequence identity to FeLV-A Env, suggesting that selection pressure in cats causes novel FeLV subgroups to emerge. PMID:26889025

  6. Novel Feline Leukemia Virus Interference Group Based on the env Gene.

    PubMed

    Miyake, Ariko; Watanabe, Shinya; Hiratsuka, Takahiro; Ito, Jumpei; Ngo, Minh Ha; Makundi, Isaac; Kawasaki, Junna; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

    2016-05-01

    Feline leukemia virus (FeLV) subgroups have emerged in infected cats via the mutation or recombination of the env gene of subgroup A FeLV (FeLV-A), the primary virus. We report the isolation and characterization of a novel env gene, TG35-2, and report that the TG35-2 pseudotype can be categorized as a novel FeLV subgroup. The TG35-2 envelope protein displays strong sequence identity to FeLV-A Env, suggesting that selection pressure in cats causes novel FeLV subgroups to emerge. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    PubMed

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  8. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    USGS Publications Warehouse

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  9. Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts

    SciTech Connect

    Vargas, Amandine Thiery, Maxime Lafond, Julie Barbeau, Benoit

    2012-03-30

    HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3 Prime end. Promoter activity and expression of both genes were induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.

  10. Proteins of Rous-associated virus 61, an avian retrovirus: common precursor for glycoproteins gp85 and gp35 and use of pactamycin to map translational order of proteins in the gag, pol, and env genes.

    PubMed Central

    Shealy, D J; Rueckert, R R

    1978-01-01

    Cells infected by Rous-associated virus 61 (RAV-61) contained a precursor-like protein, pr90, that was specifically precipitated by antiserum directed against envelope glycoproteins, gp85 and gp35. Tryptic peptide mapping showed that pr90 contained tryptic sequences of both gp85 and gp35. Pactamycin mapping experiments indicated that the two glycoproteins are translated from the env-mRNA in the order (5') gp85--gp35. The pactamycin mapping experiments also indicated a translational order of p10--(p27, p12)--p15 for the gag proteins; this agreement with the order previously reported from tryptic mapping studies on precursor pr76 of avian myeloblastosis virus implied that the stoichiometry of the core proteins was unchanged when virions were assembled in the presence of pactamycin. The reverse transcriptase proteins, unlike those of the env and gag genes, fell on the right side of the pactamycin map. This result is in accord with the idea that most, if not all, of the reverse transcriptase protein is translated by read-through of the gag(pol) message rather than by translation of a hypothetical pol-mRNA devoted solely to synthesis of that protein. Images PMID:77910

  11. Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

    PubMed Central

    Arthur, L O; Copeland, T D; Oroszlan, S; Schochetman, G

    1982-01-01

    The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat. Images PMID:6281457

  12. Feline immunodeficiency virus env gene evolution in experimentally infected cats.

    PubMed

    Kraase, Martin; Sloan, Richard; Klein, Dieter; Logan, Nicola; McMonagle, Linda; Biek, Roman; Willett, Brian J; Hosie, Margaret J

    2010-03-15

    Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus found in cats worldwide, is studied to illuminate mechanisms of lentiviral pathogenesis and to identify key components of protective immunity. During replication, lentiviruses accumulate errors of nucleotide mis-incorporation due to the low-fidelity of reverse transcriptase and recombination between viral variants, resulting in the emergence of a complex viral "quasispecies". In patients infected with HIV-1, env sequences may vary by up to 10% and the detection of quasispecies with greater heterogeneity is associated with higher viral loads and reduced CD4+ T cell numbers [1], indicating that transmission of more complex quasispecies may lead to disease progression. However, little is known about how FIV evolves as disease progresses, or why some cats develop AIDS rapidly while disease progression is slow in others. The aim of this study was to determine whether disease progression may be governed by viral evolution and to examine the diversity of viral variants emerging following infection with an infectious molecular clone. The FIV env gene encoding the envelope glycoprotein (Env) was examined at early (12 weeks) and late (322 weeks) stages of FIV infection in two groups of cats infected experimentally with the FIV-GL8 molecular clone. Viral variants were detected within quasispecies in cats in the late stages of FIV infection that contained differing amino acid compositions in several variable loops of Env, some of which were identified as determinants of receptor usage and resistance to neutralization. Therefore these results indicate that the FIV env gene evolves during the course of infection, giving rise to variants that resist neutralization and likely lead to disease progression.

  13. Detection of immunoreactive epitopes in proteins encoded by gag, env, and pol genes of human T-lymphotropic virus type I using synthetic peptides

    SciTech Connect

    Yaroslavtseva, N.G.; Kornilaeva, G.V.; Pashkova, T.A.

    1995-10-01

    Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the p19 (gag), gp46 (env), and pol proteins (pol) of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins. 25 refs., 4 tabs.

  14. Gene envY of Escherichia coli K-12 affects thermoregulation of major porin expression.

    PubMed Central

    Lundrigan, M D; Earhart, C F

    1984-01-01

    The temperature-dependent expression of OmpF and OmpC, the major channel-forming proteins of the Escherichia coli K-12 outer membrane, was studied. In wild-type cells, decreasing growth temperatures resulted in increased amounts of OmpF protein and correspondingly decreased quantities of OmpC protein. Bacteria deleted for the 13-min chromosomal region did not exhibit this temperature-dependent fluctuation in porin proteins. Plasmid pML22, which consists of pBR322 containing a 0.5-megadalton E. coli chromosomal DNA insert, complemented the thermoregulatory defect. The regulatory gene was named envY. In minicells, pML22 directed the synthesis of an envelope polypeptide (EnvY) having an apparent molecular weight of 25,000. The EnvY protein was synthesized in minicells in greater amounts at 27 degrees C than at 37 degrees C, and a reducing agent was necessary in the solubilization buffer for its subsequent detection on polyacrylamide gels. The results describe the initial characterization of a regulatory system which, along with proteins of the ompB operon, the cyclic AMP system, and the tolC gene product, is involved in a complex network affecting major porin expression. Images PMID:6317653

  15. Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole

    PubMed Central

    Manandhar, Surya P.; Ricarte, Florante; Cocca, Stephanie M.

    2013-01-01

    Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system. PMID:23166297

  16. Saccharomyces cerevisiae Env7 is a novel serine/threonine kinase 16-related protein kinase and negatively regulates organelle fusion at the lysosomal vacuole.

    PubMed

    Manandhar, Surya P; Ricarte, Florante; Cocca, Stephanie M; Gharakhanian, Editte

    2013-02-01

    Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.

  17. Construction and characterization of the recombinant Moloney murine leukemia viruses bearing the mouse Fv-4 env gene.

    PubMed Central

    Masuda, M; Yoshikura, H

    1990-01-01

    A nucleotide sequence of the mouse Fv-4 env gene was completed. Structural comparison revealed a close relationship of Fv-4 to the ecotropic Cas-Br-E murine leukemia virus isolated from a wild mouse in southern California. Various portions of the env gene of Moloney murine leukemia virus were replaced by the corresponding Fv-4 env sequence to construct recombinant murine leukemia virus clones. Infectivity of these recombinants was checked by the S+L- cell focus induction assay and the XC cell syncytium formation assay. Recombinants bearing the following Fv-4 env sequence retained ecotropic infectivity; the AccI-BamHI and BamHI-BalI regions coding for the N- and C-terminal halves of Fv-4 gp70SU, respectively; and the BalI-NcoI region encoding the cleavage site between gp70SU and p15(E)TM of the Fv-4 env. However, when the Fv-4 sequence was substituted for the p15(E)TM-coding NcoI-EcoRV region or the AccI-EcoRV region covering almost the entire env gene, infectivity was undetectable in our assays. The recombinant clone containing the Fv-4 AccI-EcoRV region, i.e., almost the entire Fv-4 env sequence, was introduced with pSV2neo into NIH 3T3 cells, and a G418r cell line named NIH(Fv4)-2 was isolated. The NIH(Fv4)-2 cell released viral particles that contained reverse transcriptase, Fv-4 env molecules as well as the other viral proteins, and viral genomic RNA. However, proviral DNA synthesis was not detected upon inoculation of this virus in NIH 3T3 cells. The loss of infectivity of the recombinant virus bearing the Fv-4 AccI-EcoRV region appeared to be caused by failure in an early step of replication. Images PMID:2304138

  18. Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.

    PubMed Central

    Mergia, A; Shaw, K E; Lackner, J E; Luciw, P A

    1990-01-01

    We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related. PMID:2152825

  19. Activation of HERV-K Env protein is essential for tumorigenesis and metastasis of breast cancer cells.

    PubMed

    Zhou, Fuling; Li, Ming; Wei, Yongchang; Lin, Kevin; Lu, Yue; Shen, Jianjun; Johanning, Gary L; Wang-Johanning, Feng

    2016-12-20

    Human endogenous retrovirus type K (HERV-K) Env protein was previously demonstrated to be overexpressed in human breast cancer (BC) cells and tissues. However, the molecular pathways driving the specific alterations are unknown. We now show that knockdown of its expression with an shRNA (shRNAenv) blocked BC cell proliferation, migration, and invasion. shRNAenv transduction also attenuated the ability of BC cells to form tumors, and notably prevented metastasis. Mechanistically, downregulation of HERV-K blocked expression of tumor-associated genes that included Ras, p-RSK, and p-ERK. The major upstream regulators influenced by HERV-K knockdown were p53, TGF- β1, and MYC. Of interest, when the HERV-K env gene was overexpressed in shRNAenv-transduced BC cells using an HERV-K env expression vector, Ras/Raf/MEK/ERK pathway signaling was restored. CDK5, which alters p53 phosphorylation in some cancers, was upregulated and p53 was downregulated when HERV-K was overexpressed. CDK5 is also a mediator of TGF-β1-induced epithelial-mesenchymal transition and migration in cancer cells, and is involved in tumor formation. Importantly, reductions in migration, invasion, and transformation of BC cells stably transduced with shRNAenv was reversed after adding back a vector with a synonymous mutation of HERV-K env. Taken together, these results indicate that HERV-K Env protein plays an important role in tumorigenesis and metastasis of BC.

  20. A novel gene that interferes with the phosphotransfer signal transduction mediated by the EnvZ osmosensor in Escherichia coli.

    PubMed

    Hirokawa, K; Ogino, T; Aiba, H; Mizuno, T

    1996-10-01

    In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli. A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes. In this study, an E. coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E. coli cells by a multicopy plasmid. The gene product was identified as F107, which was previously characterized as a hypothetical protein in E. coli genome databases. F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III. The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants. These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase. Possible mechanisms by which F107 affects the EnvZ function are discussed.

  1. env Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes

    PubMed Central

    Fujita, Donald J.; Tal, Jacov; Varmus, Harold E.; Bishop, J. Michael

    1978-01-01

    The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNAgp) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNAgp and chicken DNA and also between cDNAgp and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNAgp did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin. PMID:212576

  2. Characterisation of env and gag gene fragments of bovine leukemia viruses (BLVs) from cattle in Turkey.

    PubMed

    Alkan, Feray; Oğuzoğlu, Tuba Çiğdem; Timurkan, Mehmet Ozkan; Karapınar, Zeynep

    2011-10-01

    The aim of this work was to investigate the molecular characteristics of bovine leukemia viruses (BLVs) in Turkey. The variability of env and gag fragments of BLVs was examined using DNA from blood samples obtained for sequence analysis of BLVs in four cattle herds from three different geographical areas in Turkey. The env gene sequences were highly similar to those of Brasilian, Argentine, and Japanese BLV strains, while gag genes from Turkish BLV isolates showed greatest similarity to those of Iranian isolates. This paper is the first report on the partial characterisation of env and gag genetic fragments of BLVs from Turkey.

  3. Phenethyl Alcohol as a Suppressor of the EnvA Phenotype Associated with the envA Gene in Escherichia coli K-12

    PubMed Central

    Normark, Staffan

    1971-01-01

    In Escherichia coli K-12 the envA gene was previously shown to mediate chain formation and a decreased tolerance to several antibacterial agents. Phenethyl alcohol at low concentrations has now been found to increase the tolerance to actinomycin D, ampicillin, rifampin, and gentian violet in strains containing envA. The increased tolerance to gentian violet was correlated to a decreased uptake of the dye. A phenotype suppression of chain formation and colony morphology in envA mutants was also obtained. Except for an increase in palmitic acid, chemical analysis revealed no differences between an envA and its wild-type strain in the lipopolysaccharide part of the envelope. However, a decrease in the amount of phosphatidylglycerol and a C18: 1 fatty acid was observed in the extractable lipids of a strain containing envA. Growth in the presence of phenethyl alcohol reversed the changes in fatty acid and the phospholipid composition. Phenethyl alcohol was found to cause an immediate but transient inhibition of ribonucleic acid synthesis. It is suggested that this inhibition affects the penetrability barrier of the outer cell envelope layers in strains containing envA. Images PMID:4941568

  4. Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

    PubMed Central

    Majors, J E; Varmus, H E

    1983-01-01

    To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein. PMID:6312081

  5. Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses.

    PubMed

    Sandrin, Virginie; Muriaux, Delphine; Darlix, Jean-Luc; Cosset, François-Loïc

    2004-07-01

    Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.

  6. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  7. Role of Virus Receptor Hyal2 in Oncogenic Transformation of Rodent Fibroblasts by Sheep Betaretrovirus Env Proteins

    PubMed Central

    Liu, Shan-Lu; Duh, Fuh-Mei; Lerman, Michael I.; Miller, A. Dusty

    2003-01-01

    The ovine betaretroviruses jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause contagious cancers in the lungs and upper airways of sheep and goats. Oncogenic transformation assays using mouse and rat fibroblasts have localized the transforming activity to the Env proteins encoded by these viruses, which require the putative lung and breast cancer tumor suppressor hyaluronidase 2 (Hyal2) to promote virus entry into cells. These results suggested the hypothesis that the JSRV and ENTV Env proteins cause cancer by inhibiting the tumor suppressor activity of Hyal2. Consistent with this hypothesis, we show that human Hyal2 and other Hyal2 orthologs that can promote virus entry, including rat Hyal2, can suppress transformation by the Env proteins of JSRV and ENTV. Furthermore, we provide direct evidence for binding of the surface (SU) region of JSRV Env to human and rat Hyal2. However, mouse Hyal2 did not mediate entry of virions bearing JSRV or ENTV Env proteins, bound JSRV SU poorly if at all, and did not suppress transformation by the JSRV or ENTV Env proteins, indicating that mouse Hyal2 plays no role in transformation of mouse fibroblasts and that the Env proteins can transform at least some cells by a Hyal2-independent mechanism. Expression of human Hyal2 in mouse cells expressing JSRV Env caused a marked reduction in Env protein levels, indicating that human Hyal2 suppresses Env-mediated transformation in mouse cells by increasing Env degradation rather than by exerting a more general Env-independent tumor suppressor activity. PMID:12584308

  8. [VLP vaccines and effects of HIV-1 Env protein modifications on their antigenic properties].

    PubMed

    Vzorov, A N; Compans, R W

    2016-01-01

    An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.

  9. Coexpression of the simian immunodeficiency virus Env and Rev proteins by a recombinant human adenovirus host range mutant.

    PubMed Central

    Cheng, S M; Lee, S G; Ronchetti-Blume, M; Virk, K P; Mizutani, S; Eichberg, J W; Davis, A; Hung, P P; Hirsch, V M; Chanock, R M

    1992-01-01

    Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficiently replicates in both human and monkey cells was used to bypass the usual host range restriction of Ad infection. In addition, we show that the SIV rev gene is properly spliced from a single SIV subgenomic DNA fragment and that the Rev protein is expressed in recombinant Ad-SIV-infected human as well as monkey cells. The expression of SIV gene products in suitable live Ad vectors provides an excellent system for studying the regulation of SIV gene expression in cultured cells and evaluating the immunogenicity and protective efficacy of SIV proteins in macaques. Images PMID:1404612

  10. Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits.

    PubMed

    Yasmeen, Anila; Ringe, Rajesh; Derking, Ronald; Cupo, Albert; Julien, Jean-Philippe; Burton, Dennis R; Ward, Andrew B; Wilson, Ian A; Sanders, Rogier W; Moore, John P; Klasse, Per Johan

    2014-05-29

    The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus

  11. Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits

    PubMed Central

    2014-01-01

    Background The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T

  12. Implication of the env Gene of the Human Endogenous Retrovirus W Family in the Expression of BDNF and DRD3 and Development of Recent-Onset Schizophrenia

    PubMed Central

    Huang, WenJie; Li, Shan; Hu, YuanMing; Yu, Honglian; Luo, Feng; Zhang, Qi; Zhu, Fan

    2011-01-01

    Objective: Retrovirus has been suggested as one of agents involved in the development of schizophrenia. In the present study, we examined the role of the human endogenous retrovirus W family (HERV-W) env gene in the etiopathogenesis of recent-onset schizophrenia, using molecular and epidemiological approaches. Methods: Nested RT-PCR was used to detect the messenger RNA (mRNA) of the HERV-w env gene in plasmas. Quantitative real-time polymerase chain reaction (PCR) was employed to detect the viral reverse transcriptase activity in human sera. Human U251 glioma cells were used to study the potential role of the HERV-W env gene in the etiopathogenesis of recent-onset schizophrenia. Results: We identified genes with mRNA sequences homologous to HERV-W env gene from plasmas of 42 out of 118 individuals with recent-onset schizophrenia but not from any of 106 normal persons (P < .01, t test). Quantitative real-time PCR showed a significantly increase in the reverse transcriptase activity in the sera of patients (by 35.59%) compared with controls (by 2.83%) (P < .05, t test). Overexpression of HERV-w env in human U251 glioma cells upregulated brain-derived neurotrophic factor (BDNF), an important schizophrenia-associated gene, neurotrophic tyrosine kinase receptor type 2 (NTRK2, also called TrkB), and dopamine receptor D3 and increased the phosphorylation of cyclic adenosine monophosphate response element–binding (CREB) protein. BDNF promoter reporter gene assays showed that the HERV-W env triggers BDNF production in human U251 glioma cells. Using gene knockdown, we found that CREB is required for the expression of BDNF that is regulated by env. Conclusion: Our data revealed that the transcriptional activation of HERV is associated with the development of schizophrenia in some patients and indicated that HERV-W env regulates the expression of schizophrenia-associated genes. This report is the first to elucidate the signaling pathway responsible for the upregulation of

  13. Env7p Associates with the Golgin Protein Imh1 at the trans-Golgi Network in Candida albicans

    PubMed Central

    Rao, Kongara Hanumantha; Ghosh, Swagata

    2016-01-01

    ABSTRACT Vesicular dynamics is one of the very important aspects of cellular physiology, an imbalance of which leads to the disorders or diseases in higher eukaryotes. We report the functional characterization of a palmitoylated protein kinase from Candida albicans whose homologue in Saccharomyces cerevisiae has been reported to be involved in negative regulation of membrane fusion and was named Env7. However, the downstream target of this protein remains to be identified. Env7 in C. albicans (CaEnv7) could be isolated from the membrane fraction and localized to vesicular structures associated with the Golgi apparatus. Our work reports Env7 in C. albicans as a new player involved in maintaining the functional dynamics at the trans-Golgi network (TGN) by interacting with two other TGN-resident proteins, namely, Imh1p and Arl1p. Direct interaction could be detected between Env7p and the golgin protein Imh1p. Env7 is itself phosphorylated (Env7p) and phosphorylates Imh1 in vivo. An interaction between Env7 and Imh1 is required for the targeted localization of Imh1. CaEnv7 has a putative palmitoylation site toward both N and C termini. An N-terminal palmitoylation-defective strain retains its ability to phosphorylate Imh1 in vitro. An ENV7 homozygous mutant showed compromised filamentation in solid media and attenuated virulence, whereas an overexpressed strain affected cell wall integrity. Thus, Env7 plays a subtle but important role at the level of multitier regulation that exists at the TGN. IMPORTANCE A multitier regulation exists at the trans-Golgi network in all higher organisms. We report a palmitoylated protein kinase, Env7, that functions at the TGN interface by interacting with two more TGN-resident proteins, namely, Imh1 and Arl1. Palmitoylation seems to be important for the specific localization. This study focuses on the involvement of a ubiquitous protein kinase, whose substrates had not yet been reported from any organism, as an upstream signaling

  14. Comprehensive Characterization of the Transmitted/Founder env Genes From a Single MSM Cohort in China.

    PubMed

    Chen, Yue; Li, Ning; Zhang, Tong; Huang, Xiaojie; Cai, Fangping; Vandergrift, Nathan; Xin, Ruolei; Meng, Zhefeng; Zhang, Xiaoyan; Jiang, Chunlai; Xu, Xiaoning; Montefiori, David C; Gao, Feng; Wu, Hao

    2015-08-01

    The men having sex with men (MSM) population has become one of the major risk groups for HIV-1 infection in China. However, the epidemiological patterns, function of the env genes, and autologous and heterologous neutralization activity in the same MSM population have not been systematically characterized. The env gene sequences were obtained by the single genome amplification. The time to the most recent common ancestor was estimated for each genotype using the Bayesian Markov Chain Monte Carlo approach. Coreceptor usage was determined in NP-2 cells. Neutralization was analyzed using Env pseudoviruses in TZM-bl cells. We have obtained 547 full-length env gene sequences by single genome amplification from 30 acute/early HIV-1--infected individuals in the Beijing MSM cohort. Three genotypes (subtype B, CRF01_AE, and CRF07_BC) were identified and 20% of the individuals were infected with multiple transmitted/founder (T/F) viruses. The tight clusters of the MSM sequences regardless of geographic origins indicated nearly exclusive transmission within the MSM population and limited number of introductions. The time to the most recent common ancestor for each genotype was 10-15 years after each was first introduced in China. Disparate preferences for coreceptor usages among 3 genotypes might lead to the changes in percentage of different genotypes in the MSM population over time. The genotype-matched and genotype-mismatched neutralization activity varied among the 3 genotypes. The identification of unique characteristics for transmission, coreceptor usage, neutralization profile, and epidemic patterns of HIV-1 is critical for the better understanding of transmission mechanisms, development of preventive strategies, and evaluation of vaccine efficacy in the MSM population in China.

  15. Comprehensive Characterization of the Transmitted/founder env Genes from a Single MSM Cohort in China

    PubMed Central

    Chen, Yue; Li, Ning; Zhang, Tong; Huang, Xiaojie; Cai, Fangping; Vandergrift, Nathan; Xin, Ruolei; Meng, Zhefeng; Zhang, Xiaoyan; Jiang, Chunlai; Xu, Xiaoning; Montefiori, David C; Gao, Feng; Wu, Hao

    2015-01-01

    Background The men having sex with men (MSM) population has become one of major risk groups for HIV-1 infection in China. However, the epidemiological patterns, function of the env genes, and autologous and heterologous neutralization activity in the same MSM population have not been systematically characterized. Methods The env gene sequences were obtained by the single genome amplification (SGA). The time to the most recent common ancestor (tMRCA) was estimated for each genotype using the Bayesian MCMC approach. Coreceptor usage was determined in NP-2 cells. Neutralization was analyzed using Env pseudoviruses in TZM-bl cells. Results We have obtained 547 full-length env gene sequences by SGA from 30 acute/early HIV-1-infected individuals in the Beijing MSM cohort. Three genotypes (Subtype B, CRF01_AE, and CRF07_BC) were identified and 20% of the individuals were infected with multiple transmitted/founder (T/F) viruses. The tight clusters of the MSM sequences regardless of geographic origins indicated nearly exclusive transmission within the MSM population and limited number of introductions. The tMRCA for each genotype was 10-15 years after each was first introduced in China. Disparate preferences for coreceptor usages among three genotypes might lead to the changes in percentage of different genotypes in the MSM population over time. The genotype-matched and -mismatched neutralization activity varied among the three genotypes. Conclusions Identification of unique characteristics for transmission, coreceptor usage, neutralization profile and epidemic patterns of HIV-1 is critical for the better understanding of transmission mechanisms, development of preventive strategies, and evaluation of vaccine efficacy in the MSM population in China. PMID:25886933

  16. Erythropoietin receptor (EpoR)-dependent mitogenicity of spleen focus-forming virus correlates with viral pathogenicity and processing of env protein but not with formation of gp52-EpoR complexes in the endoplasmic reticulum.

    PubMed Central

    Wang, Y; Kayman, S C; Li, J P; Pinter, A

    1993-01-01

    Recent evidence suggests that interactions between spleen focus-forming virus (SFFV) env products and the erythropoietin receptor (EpoR) are responsible for viral pathogenicity. Infection of factor-dependent cell lines expressing epoR (the cloned gene for EpoR) with SFFVP is mitogenic, generating cell lines that are no longer dependent on added growth factor, and an immunoprecipitable complex between EpoR and immature env protein in the endoplasmic reticulum has been identified. The dependence of these in vitro activities on env protein processing and their relationship to pathogenicity of SFFV were explored by using glycosylation site mutants of SFFV env. Mutants carrying Asn-->Asp mutations at each of the two consensus signals for N-linked glycosylation in the N-terminal domain of SFFVAP-L env (gs1 and gs2), the gs1-2- double mutant, and the gs0 quadruple mutant (mutated at all four signals utilized for N-linked glycosylation in SFFVAP-L env) were made. The primary translation products (gp52) of single-site mutant envs were processed into more highly glycosylated forms, and the corresponding viruses induced splenomegaly in susceptible mice, whereas the gs1-2- and gs0 proteins were not processed, and these viruses were not pathogenic. Unprocessed env proteins of both pathogenic and nonpathogenic mutants coprecipitated with EpoR. In the BaF3 cell assay for epoR-dependent mitogenicity, the pathogenic single mutants induced factor-independent growth efficiently whereas the nonpathogenic gs1-2- and gs0 mutants did not. These data demonstrate that the ability of gp52 to form complexes with EpoR in the endoplasmic reticulum is not sufficient for either mitogenicity in cell culture or induction of splenomegaly in mice while supporting the hypothesis that pathogenicity and mitogenicity of SFFV both result from an interaction between EpoR and SFFV env protein. Images PMID:8437218

  17. env Gene typing of human immunodeficiency virus type 1 strains on electronic microarrays.

    PubMed

    Saunders, N A; Alexander, S; Tatt, I

    2005-04-01

    The NanoChip system was used for subtyping human immunodeficiency virus type 1 (HIV-1) strains using probes complementary to the V1 region of the env gene. Probes for six subtypes (A to D, F, and G) and two circulating recombinant forms (AG and AE) of HIV-1 group M were included. The specificity of these oligonucleotides had been evaluated previously in a DNA enzyme immunoassay. Samples from 112 patient sera were used as templates in a nested reverse transcription-PCR to produce amplicons that were applied to the array. The array was then hybridized successively to pairs of oligonucleotide probes. The strains were assigned a subtype on the basis of their probe hybridization patterns. One strain gave a contradictory pattern and was designated as untypeable by the NanoChip assay. Eighty-eight strains gave hybridization patterns that allowed a correct subtype designation to be made by the NanoChip assay compared to either the sequence or the heteroduplex mobility assay (HMA)-determined subtypes. Thirteen strains that reacted with the subtype A probe (SA2) were incorrectly assigned to subtype A, or to one of the related circulating recombinant types (AE or AG), on the basis of reactions with probe SAE1 or SAG1. The results indicate that these oligonucleotides have relatively low specificities. The probe subtypes of three strains matched the subtypes determined for the gag and pol genes but not the env gene, suggesting that a recombination event may have occurred within the env gene. Overall, the NanoChip assay gave results comparable to those for HMA and sequencing and provides a convenient and cost-effective means by which to subtype HIV-1.

  18. env Gene Typing of Human Immunodeficiency Virus Type 1 Strains on Electronic Microarrays

    PubMed Central

    Saunders, N. A.; Alexander, S.; Tatt, I.

    2005-01-01

    The NanoChip system was used for subtyping human immunodeficiency virus type 1 (HIV-1) strains using probes complementary to the V1 region of the env gene. Probes for six subtypes (A to D, F, and G) and two circulating recombinant forms (AG and AE) of HIV-1 group M were included. The specificity of these oligonucleotides had been evaluated previously in a DNA enzyme immunoassay. Samples from 112 patient sera were used as templates in a nested reverse transcription-PCR to produce amplicons that were applied to the array. The array was then hybridized successively to pairs of oligonucleotide probes. The strains were assigned a subtype on the basis of their probe hybridization patterns. One strain gave a contradictory pattern and was designated as untypeable by the NanoChip assay. Eighty-eight strains gave hybridization patterns that allowed a correct subtype designation to be made by the NanoChip assay compared to either the sequence or the heteroduplex mobility assay (HMA)-determined subtypes. Thirteen strains that reacted with the subtype A probe (SA2) were incorrectly assigned to subtype A, or to one of the related circulating recombinant types (AE or AG), on the basis of reactions with probe SAE1 or SAG1. The results indicate that these oligonucleotides have relatively low specificities. The probe subtypes of three strains matched the subtypes determined for the gag and pol genes but not the env gene, suggesting that a recombination event may have occurred within the env gene. Overall, the NanoChip assay gave results comparable to those for HMA and sequencing and provides a convenient and cost-effective means by which to subtype HIV-1. PMID:15815017

  19. Phylogenetic and structural diversity in the feline leukemia virus env gene.

    PubMed

    Watanabe, Shinya; Kawamura, Maki; Odahara, Yuka; Anai, Yukari; Ochi, Haruyo; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

    2013-01-01

    Feline leukemia virus (FeLV) belongs to the genus Gammaretrovirus, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral env sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV env genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1-7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV env gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated de novo in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats.

  20. Adenovirus prime, Env protein boost vaccine protects against neutralization-resistant SIVsmE660 variants in rhesus monkeys.

    PubMed

    Keele, Brandon F; Li, Wenjun; Borducchi, Erica N; Nkolola, Joseph P; Abbink, Peter; Chen, Bing; Seaman, Michael S; Barouch, Dan H

    2017-06-05

    Previous studies have shown that DNA prime, Ad5 boost vaccines protect against neutralization-sensitive but not neutralization-resistant virus variants within the SIVsmE660 swarm. Here we show that Ad prime, Env protein boost vaccines protect against neutralization-resistant SIVsmE660 variants. We perform two studies in rhesus monkeys with Ad35/Ad26 vectors expressing SIVmac239 Gag/Pol/Env with or without an AS01B-adjuvanted SIVmac32H gp140 protein boost. In a repetitive, low-dose challenge study, we observe robust protection against acquisition of infection by both Ad Alone and Ad/Env vaccines. In a single, high-dose challenge study, only the Ad/Env vaccine affords significant protection against acquisition of infection. Analysis of transmitted/founder (T/F) viruses from this study demonstrates that the Ad/Env vaccine blocks both neutralization-sensitive and neutralization-resistant SIVsmE660 variants in rhesus monkeys with restrictive TRIM5α alleles. These data demonstrate that the adjuvanted Env protein boost is critical for protecting against high-dose SIVsmE660 challenge and for blocking neutralization-resistant viruses within the SIVsmE660 swarm.

  1. Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene.

    PubMed

    Pelisson, A; Mejlumian, L; Robert, V; Terzian, C; Bucheton, A

    2002-10-01

    The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.

  2. Avian hemangioma retrovirus induces cell proliferation via the envelope (env) gene.

    PubMed

    Alian, A; Sela-Donenfeld, D; Panet, A; Eldor, A

    2000-10-10

    Several years ago, a field strain retrovirus, avian hemangioma virus (AHV), was isolated from hemangioma tumors in layer hens. Sequence analysis indicated that the AHV genome contains the three prototypic retroviral genes, gag, pol, and env, and is devoid of an oncogene. In cultured endothelial cells, however, AHV induced a significant cytopathic effect through a typical apoptotic cascade. We now demonstrate that AHV also induces cell proliferation and anchorage-independent growth of BSC-1 epithelial cells and NIH-3T3 fibroblasts. This was shown by measurements of (1) cell viability, (2) DNA synthesis, (3) flow cytometry analysis of the cell DNA content, and (4) clonogenic efficiency of the infected cells. Anchorage-independent cell growth was demonstrated by colony formation in soft agar. Moreover, the AHV env gene was cloned into a MuLV-based retroviral vector, and infection of NIH-3T3 cells with this vector induced cell proliferation as well as clonogenic growth. These results suggest that AHV, which is devoid of an oncogene, is a pleiotropic activator capable of inducing either apoptosis or cellular proliferation, depending on the infected cell type. Copyright 2000 Academic Press.

  3. Ultrastructural evidence of an interaction between Env and Gag proteins during assembly of HIV type 1.

    PubMed

    Bugelski, P J; Maleeff, B E; Klinkner, A M; Ventre, J; Hart, T K

    1995-01-01

    Assembly and budding of retroviruses is believed to involve a complex interaction of envelope and capsid proteins at the host cell membrane. The nature of these interactions is, however, incompletely understood. Studies of the topography of the surface of HIV-1 have shown that the envelope glycoprotein projections (knobs) are arranged in a T = 7 levo rotational symmetry. Similarly, an icosahedral structure has been suggested for the p17 matrix of HIV-1. In an effort to investigate whether there is a structural interaction between these molecules, virions whose maturation was blocked by an inhibitor of HIV protease were studied using cytochemistry, morphometry, and 2D fast Fourier transform image enhancement. Analysis of the relationship between core morphology and the topographic distribution of envelope glycoprotein projections on HIV-1 provided structural evidence of an interaction between Env and Gag proteins. Furthermore, image enhancement revealed a periodic substructure in the Pr55gag plaque. Taken together, the data suggest an interaction between Pr55gag and the gp120-gp41 complex during assembly and budding of HIV-1. This interaction may, in part, contribute to determining the amount of Env glycoprotein that will be incorporated into a virion, and therefore play a role in the biology of HIV-1.

  4. A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins.

    PubMed Central

    Benko, D M; Schwartz, S; Pavlakis, G N; Felber, B K

    1990-01-01

    We have characterized a novel 28-kilodalton protein, p28tev, detected in human immunodeficiency virus type 1-infected cells. tev is recognized by both tat and rev monospecific antibodies. tev is initiated at the tat AUG and contains the first exon of tat at its amino terminus, a small portion of env in the middle, and the second exon of rev at its carboxy terminus. A cDNA clone producing tev was cloned and expressed in human cells. Sequence analysis revealed that the tev mRNA is generated by splicing to a novel exon located in the env region. This identifies a fourth class of multiply spliced human immunodeficiency virus mRNAs, produced in infected and transfected cells. tev is regulated during the virus life cycle similarly to the other regulatory proteins, tat, rev, and nef, and displays both tat and rev activities in functional assays. Since tev contains important functional domains of tat and rev and is produced very early after transfection, it may be an important regulator in the initial phase of virus expression. Another rev-related protein, p18(6)Drev, containing env and rev sequences, was characterized and was found not to have detectable rev activity. Images PMID:2186172

  5. Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation

    PubMed Central

    Liu, Shan-Lu; Lerman, Michael I.; Miller, A. Dusty

    2003-01-01

    Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway. PMID:12829832

  6. Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology.

    PubMed

    Hantson, Anke; Fikkert, Valery; Van Remoortel, Barbara; Pannecouque, Chistophe; Cherepanov, Peter; Matthews, Barry; Holan, George; De Clercq, Erik; Vandamme, Anne-Mieke; Debyser, Zeger; Witvrouw, Myriam

    2005-01-01

    A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3/SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3/SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3/SPL2923 towards DS was fully reproduced following gp160-recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resistance to enfuvirtide. Unexpectedly, the reduced sensitivity towards SPL2923 was not fully reproduced after gp160-recombination. The search for mutations in NL4.3/SPL2923 in viral genes other than env revealed several mutations in the gene encoding the HIV p17 matrix protein (MA) and one mutation in the gene encoding the p24 capsid protein (CA). In order to analyse the impact of the gag mutations alone and in combination with the mutations in env on the phenotypic resistance towards SPL2923, we developed a novel p17- and p17/gp160-CVT. Phenotypic analysis of the NL4.3/SPL2923 p17- and p17/gp160-recombined strains indicated that the mutations in both env and gag have to be present to fully reproduce the resistance of NL4.3/SPL2923 towards SPL2923.

  7. Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus.

    PubMed

    Bęczkowski, Paweł M; Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J; Hosie, Margaret J

    2015-04-01

    Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10(-3) substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. © 2015 The Authors.

  8. Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus

    PubMed Central

    Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J.; Hosie, Margaret J.

    2015-01-01

    Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10−3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3–V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3–V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. PMID:25535323

  9. The 17 Nucleotides Downstream from the env Gene Stop Codon Are Important for Murine Leukemia Virus Packaging

    PubMed Central

    Shin Yu, Seung; Kim, Jong-Mook; Kim, Sunyoung

    2000-01-01

    We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of the env gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5′ long terminal repeat (LTR) and the other immediately upstream from the 3′ LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed. PMID:10954583

  10. Effect of point mutations in the N terminus of the lentivirus lytic peptide-1 sequence of human immunodeficiency virus type 1 transmembrane protein gp41 on Env stability.

    PubMed

    Lee, Sheau-Fen; Ko, Chiung-Yuan; Wang, Chin-Tien; Chen, Steve S-L

    2002-05-03

    To understand the role of the lentivirus lytic peptide-1 region of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp) 41 in viral infection, we examined the effects on virus replication of single amino acid deletions spanning this region in an infectious provirus of the HXB2 strain. Among the mutants analyzed, only the deletion of one of the two adjacent valine residues located at positions 832 and 833 (termed the Delta 833 mutant for simplicity) greatly reduced the steady-state, cell-associated levels of the Env precursor and gp120, as opposed to the wild-type virus. The altered Env phenotype resulted in severely impaired virus infectivity and gp120 incorporation into this mutant virion. Analyses of additional mutants with deletions at Ile-830, Ala-836, and Ile-840 demonstrated that the Delta 830 mutant exhibited the most significant inhibitory effect on Env steady-state expression. These results indicate that the N terminus of the lentivirus lytic peptide-1 region is critical for Env steady-state expression. Among the mutant viruses encoding Env proteins in which residues Val-832 and Val-833 were individually substituted by nonconserved amino acids Ala, Ser, or Pro, which were expected to disrupt the alpha-helical structure in the increasingly severe manner of Pro > Ser > Ala, only the 833P mutant exhibited significantly reduced steady-state Env expression. Pulse labeling and pulse-chase studies demonstrated that the Delta 830, Delta 833, and 833P mutants of Env proteins degraded more rapidly in a time-dependent manner after biosynthesis than did the wild-type Env. The results indicate that residue 830 and 833 mutations are likely to induce a conformational change in Env that targets the mutant protein for cellular degradation. Our study has implications about the structural determinants located at the N terminus of the lentivirus lytic peptide-1 sequence of gp41 that affect the fate of Env in virus-infected cells.

  11. Co-regulation of polysaccharide production, motility, and expression of type III secretion genes by EnvZ/OmpR and GrrS/GrrA systems in Erwinia amylovora.

    PubMed

    Li, Wenting; Ancona, Veronica; Zhao, Youfu

    2014-02-01

    The EnvZ/OmpR and GrrS/GrrA systems, two widely distributed two-component systems in gamma-Proteobacteria, negatively control amylovoran biosynthesis in Erwinia amylovora, and the two systems regulate motility in an opposing manner. In this study, we examined the interplay of EnvZ/OmpR and GrrS/GrrA systems in controlling various virulence traits in E. amylovora. Results showed that amylovoran production was significantly higher when both systems were inactivated, indicating that the two systems act as negative regulators and their combined effect on amylovoran production appears to be enhanced. In contrast, reduced motility was observed when both systems were deleted as compared to that of grrA/grrS mutants and WT strain, indicating that the two systems antagonistically regulate motility in E. amylovora. In addition, glycogen accumulation was much higher in envZ/ompR and two triple mutants than that of grrS/grrA mutants and WT strain, suggesting that EnvZ/OmpR plays a dominant role in regulating glycogen accumulation, whereas levan production was significantly lower in the grrS/grrA and two triple mutants as compared with that of WT and envZ/ompR mutants, indicating that GrrS/GrrA system dominantly controls levan production. Furthermore, both systems negatively regulated expression of three type III secretion (T3SS) genes and their combined negative effect on hrp-T3SS gene expression increased when both systems were deleted. These results demonstrated that EnvZ/OmpR and GrrS/GrrA systems co-regulate various virulence factors in E. amylovora by still unknown mechanisms or through different target genes, sRNAs, or proteins, indicating that a complex regulatory network may be involved, which needs to be further explored.

  12. A machine learning approach for identifying amino acid signatures in the HIV env gene predictive of dementia.

    PubMed

    Holman, Alexander G; Gabuzda, Dana

    2012-01-01

    The identification of nucleotide sequence variations in viral pathogens linked to disease and clinical outcomes is important for developing vaccines and therapies. However, identifying these genetic variations in rapidly evolving pathogens adapting to selection pressures unique to each host presents several challenges. Machine learning tools provide new opportunities to address these challenges. In HIV infection, virus replicating within the brain causes HIV-associated dementia (HAD) and milder forms of neurocognitive impairment in 20-30% of patients with unsuppressed viremia. HIV neurotropism is primarily determined by the viral envelope (env) gene. To identify amino acid signatures in the HIV env gene predictive of HAD, we developed a machine learning pipeline using the PART rule-learning algorithm and C4.5 decision tree inducer to train a classifier on a meta-dataset (n = 860 env sequences from 78 patients: 40 HAD, 38 non-HAD). To increase the flexibility and biological relevance of our analysis, we included 4 numeric factors describing amino acid hydrophobicity, polarity, bulkiness, and charge, in addition to amino acid identities. The classifier had 75% predictive accuracy in leave-one-out cross-validation, and identified 5 signatures associated with HAD diagnosis (p<0.05, Fisher's exact test). These HAD signatures were found in the majority of brain sequences from 8 of 10 HAD patients from an independent cohort. Additionally, 2 HAD signatures were validated against env sequences from CSF of a second independent cohort. This analysis provides insight into viral genetic determinants associated with HAD, and develops novel methods for applying machine learning tools to analyze the genetics of rapidly evolving pathogens.

  13. A Machine Learning Approach for Identifying Amino Acid Signatures in the HIV Env Gene Predictive of Dementia

    PubMed Central

    Holman, Alexander G.; Gabuzda, Dana

    2012-01-01

    The identification of nucleotide sequence variations in viral pathogens linked to disease and clinical outcomes is important for developing vaccines and therapies. However, identifying these genetic variations in rapidly evolving pathogens adapting to selection pressures unique to each host presents several challenges. Machine learning tools provide new opportunities to address these challenges. In HIV infection, virus replicating within the brain causes HIV-associated dementia (HAD) and milder forms of neurocognitive impairment in 20–30% of patients with unsuppressed viremia. HIV neurotropism is primarily determined by the viral envelope (env) gene. To identify amino acid signatures in the HIV env gene predictive of HAD, we developed a machine learning pipeline using the PART rule-learning algorithm and C4.5 decision tree inducer to train a classifier on a meta-dataset (n = 860 env sequences from 78 patients: 40 HAD, 38 non-HAD). To increase the flexibility and biological relevance of our analysis, we included 4 numeric factors describing amino acid hydrophobicity, polarity, bulkiness, and charge, in addition to amino acid identities. The classifier had 75% predictive accuracy in leave-one-out cross-validation, and identified 5 signatures associated with HAD diagnosis (p<0.05, Fisher’s exact test). These HAD signatures were found in the majority of brain sequences from 8 of 10 HAD patients from an independent cohort. Additionally, 2 HAD signatures were validated against env sequences from CSF of a second independent cohort. This analysis provides insight into viral genetic determinants associated with HAD, and develops novel methods for applying machine learning tools to analyze the genetics of rapidly evolving pathogens. PMID:23166702

  14. Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes

    PubMed Central

    Machinaga, Akihito; Ishihara, Syuhei; Shirai, Akiko; Takase-Yoden, Sayaka

    2016-01-01

    Friend murine leukemia virus (MLV) belongs to the gamma retroviruses of the Retroviridae family. The positive-sense RNA of its genome contains a 5′ long terminal repeat (LTR), 5′ leader sequence, gag, pol, env, and 3′ LTR. Transcription from proviral DNA begins from the R region of the 5′ LTR and ends at the polyadenylation signal located at the R region of the other end of the 3′ LTR. There is a 5′ splice site in the 5′ leader sequence and a 3′ splice site at the 3′ end of the pol region. Both full-length unspliced mRNAs and a singly spliced mRNA (env-mRNA) are produced in MLV-infected cells. The MLV Env protein plays important roles both in viral adsorption to host cells and in neuropathogenic disease in MLV-infected mice and rats. Understanding the regulatory mechanisms controlling Env expression is important for determining the functions of the Env protein. We have previously shown that splicing increases env-mRNA stability and translation efficiency. Generally, mRNA polysome formation correlates with translation efficiency. Therefore, here we investigated the effects of env-mRNA splicing on polysome formation to identify mechanisms for Env up-regulation due to splicing. We performed polysome profile analyses using Env-expression plasmids producing spliced or unspliced env-mRNA and showed that the former formed polysomes more efficiently than the latter. Thus, splicing of env-mRNA facilitated polysome formation, suggesting that this contributes to up-regulation of Env expression. We replaced the env region of the expression plasmids with a luciferase (luc) gene, and found that in this case both unspliced and spliced luc-mRNA formed polysomes to a similar extent. Thus, we conclude that whether mRNA polysome formation is affected by splicing depends on the structure of gene in question. PMID:26909075

  15. Nature of the Penetration Barrier in Escherichia coli K-12: Effect of Macromolecular Inhibition on Penetrability in Strains Containing the envA Gene

    PubMed Central

    Normark, Staffan; Westling, Britta

    1971-01-01

    The envA mutation in Escherichia coli K-12, which maps at 1.5 min, was previously shown to mediate sensitivity to gentian violet as well as to several antibiotics. Moreover, strains containing the envA gene were recently found to be lysed by lysozyme in the absence of ethylenediaminetetraacetate. It is here reported that the envA mutation mediates an increased uptake of gentian violet. The uptake of the dye was markedly affected by growth with different antibiotics interfering with macromolecular synthesis. Amino acid starvation of a strain containing envA with a stringent control of ribonucleic acid (RNA) synthesis resulted in a decreased uptake of gentian violet. However, no decrease in dye uptake was found during starvation in an envA transductant with a relaxed control of RNA synthesis. Inhibition of deoxyribonucleic acid (DNA) synthesis by nalidixic acid decreased the uptake of gentian violet of envA cells and, in addition, rendered the cells insensitive to the lytic action of lysozyme. Chloramphenicol treatment increased penetrability in wild-type and starved envA cells. In most instances, this effect of chloramphenicol was prevented by selectively interfering with DNA or RNA synthesis. A coordinate regulation of nucleic acid synthesis and penetrability is suggested. PMID:4941566

  16. Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics.

    PubMed

    Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

    2014-01-01

    There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to

  17. Different HIV-1 env frames: gp120 and ASP (antisense protein) biosynthesis, and theirs co-variation tropic amino acid signatures in X4- and R5-viruses.

    PubMed

    Dimonte, Salvatore

    2017-01-01

    Antisense protein (ASP) is the new actor of viral life of Human Immunodeficiency Virus type 1 (HIV-1) although proposed above 20 years ago. The asp ORF is into complementary strand of the gp120/gp41 junction of env gene. The ASP biological role remains little known. Knowing the Env markers of viral tropism, a dataset of sequences (660 strains) was used to analyze the hypothetical ASP involvement in CCR5 (R5) and/or CXCR4 (X4) co-receptor interaction. Preliminarily, prevalence of ASP and gp120V3 mutations was performed; following association among mutations were elaborate. The classical V3 tropic-signatures were confirmed, and 36 R5- and 22 X4-tropic ASP mutations were found. Moreover, by analyzing the ASP sequences, 36 out of 179 amino acid positions significantly associated with different co-receptor usage were found. Several statistically significant associations between gp120V3 and ASP mutations were observed. The dendrogram showed the existence of a cluster associated with R5-usage and a large cluster associated with X4-usage. These results show that gp120V3 and specific amino acid changes in ASP are associated together with CXCR4 and/or CCR5-usage. These findings implement previous observations on unclear ASP functions. J. Med. Virol. 89:112-122, 2017. © 2016 Wiley Periodicals, Inc.

  18. Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.

    PubMed

    Lee, Jeong Hyun; de Val, Natalia; Lyumkis, Dmitry; Ward, Andrew B

    2015-10-06

    Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.

  19. Chimeric adenovirus type 5/35 vector encoding SIV gag and HIV env genes affords protective immunity against the simian/human immunodeficiency virus in monkeys.

    PubMed

    Someya, Kenji; Xin, Ke-Qin; Ami, Yasushi; Izumi, Yasuyuki; Mizuguchi, Hiroyuki; Ohta, Shinrai; Yamamoto, Naoki; Honda, Mitsuo; Okuda, Kenji

    2007-10-25

    Replication-defective adenovirus type 5 (Ad5) vector-based vaccines are widely known to induce strong immunity against immunodeficiency viruses. To exploit this immunogenicity while overcoming the potential problem of preexisting immunity against human adenoviruses type 5, we developed a recombinant chimeric adenovirus type 5 with type 35 fiber vector (rAd5/35). We initially produced a simian immunodeficiency virus (SIV) gag DNA plasmid (rDNA-Gag), a human immunodeficiency virus type 1 (HIV-1) 89.6 env DNA plasmid (rDNA-Env) and a recombinant Ad5/35 vector encoding the SIV gag and HIV env gene (rAd5/35-Gag and rAd5/35-Env). Prime-boost vaccination with rDNA-Gag and -Env followed by high doses of rAd5/35-Gag and -Env elicited higher levels of cellular immune responses than did rDNAs or rAd5/35s alone. When challenged with a pathogenic simian human immunodeficiency virus (SHIV), animals receiving a prime-boost regimen or rAd5/35s alone maintained a higher number of CD4(+) T cells and remarkably suppressed plasma viral RNA loads. These findings suggest the clinical promise of an rAd5/35 vector-based vaccine.

  20. Variability of the env gene in cynomolgus macaques persistently infected with human immunodeficiency virus type 2 strain ben.

    PubMed Central

    Tolle, T; Petry, H; Bachmann, B; Hunsmann, G; Lüke, W

    1994-01-01

    The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection. PMID:8139054

  1. [Functional analysis of Grp and Iris, the gag and env domesticated errantivirus genes, in the Drosophila melanogaster genome].

    PubMed

    Makhnovskii, P A; Kuzmin, I V; Nefedova, L N; Kima, A I

    2016-01-01

    Drosophila melanogaster is the only invertebrate that contains endogenous retroviruses, which are called errantiviruses. Two domesticated genes, Grp and Iris, which originate from errantivirus gag and env, respectively, have been found in the D. melanogaster genome. The functions performed by the genes in Drosophila are still unclear. To identify the functions of domesticated gag and env in the D. melanogaster genome, expression of Iris and Grp was studied in strains differing by the presence or absence of the functional gypsy errantivirus. In addition, the expression levels were measured after injection of gram-positive and gram-negative bacteria, which activate different immune response pathways, and exposure to various abiotic stress factors. The presence of functional D. melanogaster retrovirus gypsy was found to increase the Grp expression level in somatic tissues of the carcass, while exerting no effect on the Iris expression level. Activation of the immune response in D. melanogaster by bacteria Bacillus cereus increased the Grp expression level and did not affect Iris expression. As for the effects of abiotic stress factors (oxidative stress, starvation, and heat and cold stress), the Grp expression level increased in response to starvation in D. melanogaster females, and the Iris expression level was downregulated in heat shock and oxidative stress. Based on the findings, Grp was assumed to play a direct role in the immune response in D. melanogaster; Iris is not involved in immune responses, but and apparently performs a cell function that is inhibited in stress.

  2. Early Steps of Jaagsiekte Sheep Retrovirus-Mediated Cell Transformation Involve the Interaction between Env and the RALBP1 Cellular Protein

    PubMed Central

    Monot, Margaux; Erny, Alexandra; Gineys, Barbara; Desloire, Sophie; Dolmazon, Christine; Aublin-Gex, Anne; Lotteau, Vincent; Archer, Fabienne

    2015-01-01

    ABSTRACT Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope. IMPORTANCE JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep. PMID:26041289

  3. The opgC gene is required for OPGs succinylation and is osmoregulated through RcsCDB and EnvZ/OmpR in the phytopathogen Dickeya dadantii

    PubMed Central

    Bontemps-Gallo, Sébastien; Madec, Edwige; Robbe-Masselot, Catherine; Souche, Erika; Dondeyne, Jacqueline; Lacroix, Jean-Marie

    2016-01-01

    Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence. PMID:26790533

  4. HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization

    PubMed Central

    Bale, Shridhar; Phad, Ganesh E.; Guenaga, Javier; Wilson, Richard; Soldemo, Martina; McKee, Krisha; Sundling, Christopher; Mascola, John; Li, Yuxing; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

    2014-01-01

    Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01. PMID:25166308

  5. A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays

    SciTech Connect

    Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Decker, Julie M.; Keele, Brandon F.; Salazar-Gonzalez, Jesus F.; Grayson, Truman; McPherson, David T.; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; Williamson, Carolyn; Shaw, George M.; Hahn, Beatrice H.

    2010-02-20

    Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01{sub A}/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

  6. Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

    PubMed Central

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D.

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. PMID:24614057

  7. Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

    PubMed

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

  8. Radiation-induced human endogenous retrovirus (HERV)-R env gene expression by epigenetic control.

    PubMed

    Lee, Ja-Rang; Ahn, Kung; Kim, Yun-Ji; Jung, Yi-Deun; Kim, Heui-Soo

    2012-11-01

    It is commonly accepted that ionizing radiation induces genomic instability by changes in genomic structure, epigenetic regulation and gene expression. Human endogenous retroviruses (HERV)-R also are often differentially expressed between normal and disease tissues under unstable genomic conditions and are implicated in the pathogenesis of several human diseases. To understand the influence of ionizing radiation on HERV-R expression, we performed quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses using γ-irradiated normal human cells. Compared to nonirradiated cells, HERV-R expression was up-regulated in γ-irradiated cells. The regulatory mechanism of HERV-R expression in irradiated cells was investigated by methylation analyses of HERV-R 5'LTRs and treatment with garcinol. These data indicated that the up-regulated transcription of HERV-R may be regulated by radiation-induced epigenetic changes induced by histone modification, and thus could be of great importance for understanding the relationship between radiation-induced biological effects and transposable elements.

  9. Retroviral env glycoprotein trafficking and incorporation into virions.

    PubMed

    Murakami, Tsutomu

    2012-01-01

    Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

  10. Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins

    PubMed Central

    Wrensch, Florian; Hoffmann, Markus; Gärtner, Sabine; Nehlmeier, Inga; Winkler, Michael

    2016-01-01

    ABSTRACT Interferon-induced transmembrane proteins (IFITMs) can inhibit the cellular entry of several enveloped viruses, including simian immunodeficiency virus (SIV). The blockade of SIV by IFITMs is isolate specific, raising the question of which parameters impact sensitivity to IFITM. We show that the virion context in which SIV-Env is presented and the efficiency of virion incorporation determine Env susceptibility to inhibition by IFITMs. Thus, determinants other than the nature of the envelope protein can impact the IFITM sensitivity of viral entry. IMPORTANCE The host cell-encoded IFITM proteins can block viral entry and are an important component of the innate defenses against viral infection. However, the determinants controlling whether a virus is susceptible to blockade by IFITM proteins are incompletely understood. Our study shows that the amount of envelope proteins incorporated into virions as well as the nature of the virion particle itself can impact the sensitivity of viral entry to IFITMs. These results show for the first time that determinants other than the viral envelope protein can impact sensitivity to IFITM and have implications for the interpretation of previously published data on inhibition of viruses by IFITM proteins. Moreover, our findings might help to define the mechanism underlying the antiviral activity of IFITM proteins. PMID:27807233

  11. Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins.

    PubMed

    Wrensch, Florian; Hoffmann, Markus; Gärtner, Sabine; Nehlmeier, Inga; Winkler, Michael; Pöhlmann, Stefan

    2017-01-15

    Interferon-induced transmembrane proteins (IFITMs) can inhibit the cellular entry of several enveloped viruses, including simian immunodeficiency virus (SIV). The blockade of SIV by IFITMs is isolate specific, raising the question of which parameters impact sensitivity to IFITM. We show that the virion context in which SIV-Env is presented and the efficiency of virion incorporation determine Env susceptibility to inhibition by IFITMs. Thus, determinants other than the nature of the envelope protein can impact the IFITM sensitivity of viral entry. The host cell-encoded IFITM proteins can block viral entry and are an important component of the innate defenses against viral infection. However, the determinants controlling whether a virus is susceptible to blockade by IFITM proteins are incompletely understood. Our study shows that the amount of envelope proteins incorporated into virions as well as the nature of the virion particle itself can impact the sensitivity of viral entry to IFITMs. These results show for the first time that determinants other than the viral envelope protein can impact sensitivity to IFITM and have implications for the interpretation of previously published data on inhibition of viruses by IFITM proteins. Moreover, our findings might help to define the mechanism underlying the antiviral activity of IFITM proteins. Copyright © 2017 Wrensch et al.

  12. Comparison of Viral Env Proteins from Acute and Chronic Infections with Subtype C Human Immunodeficiency Virus Type 1 Identifies Differences in Glycosylation and CCR5 Utilization and Suggests a New Strategy for Immunogen Design

    PubMed Central

    Ping, Li-Hua; Joseph, Sarah B.; Anderson, Jeffrey A.; Abrahams, Melissa-Rose; Salazar-Gonzalez, Jesus F.; Kincer, Laura P.; Treurnicht, Florette K.; Arney, Leslie; Ojeda, Suany; Zhang, Ming; Keys, Jessica; Potter, E. Lake; Chu, Haitao; Moore, Penny; Salazar, Maria G.; Iyer, Shilpa; Jabara, Cassandra; Kirchherr, Jennifer; Mapanje, Clement; Ngandu, Nobubelo; Seoighe, Cathal; Hoffman, Irving; Gao, Feng; Tang, Yuyang; Labranche, Celia; Lee, Benhur; Saville, Andrew; Vermeulen, Marion; Fiscus, Susan; Morris, Lynn; Karim, Salim Abdool; Haynes, Barton F.; Shaw, George M.; Korber, Bette T.; Hahn, Beatrice H.; Cohen, Myron S.; Montefiori, David; Williamson, Carolyn

    2013-01-01

    Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets. PMID:23616655

  13. Fc Receptor-Mediated Activities of Env-Specific Human Monoclonal Antibodies Generated from Volunteers Receiving the DNA Prime-Protein Boost HIV Vaccine DP6-001.

    PubMed

    Costa, Matthew R; Pollara, Justin; Edwards, Regina Whitney; Seaman, Michael S; Gorny, Miroslaw K; Montefiori, David C; Liao, Hua-Xin; Ferrari, Guido; Lu, Shan; Wang, Shixia

    2016-11-15

    HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years of infection, and therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that moderate protection is possible and that this protection may correlate with antibody-dependent cellular cytotoxicity (ADCC) activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, the DP6-001 trial, a polyvalent Env DNA prime-protein boost formulation could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report on the production and analysis of HIV-1 Env-specific human monoclonal antibodies (hMAbs) isolated from vaccinees in the DP6-001 trial. For this initial report, 13 hMAbs from four vaccinees in the DP6-001 trial showed broad binding to gp120 proteins of diverse subtypes both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc receptor-mediated functional activities, including ADCC and antibody-dependent cellular phagocytosis (ADCP) activities, were present with both immune sera and isolated MAbs, confirming the induction of nonneutralizing functional hMAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive hMAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design.

  14. The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm.

    PubMed

    Wang, Loo Chien; Morgan, Leslie K; Godakumbura, Pahan; Kenney, Linda J; Anand, Ganesh S

    2012-05-30

    Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ(c)). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His(243)). EnvZ(c) exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His(243), suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.

  15. Interrelationships of VEL1 and ENV1 in light response and development in Trichoderma reesei.

    PubMed

    Bazafkan, Hoda; Dattenböck, Christoph; Stappler, Eva; Beier, Sabrina; Schmoll, Monika

    2017-01-01

    Sexual development is regulated by a complex regulatory mechanism in fungi. For Trichoderma reesei, the light response pathway was shown to impact sexual development, particularly through the photoreceptor ENVOY. Moreover, T. reesei communicates chemically with a potential mating partner in its vicinity, a response which is mediated by the velvet family protein VEL1 and its impact on secondary metabolism. We therefore studied the regulatory interactions of ENV1 and VEL1 with a focus on sexual development. Although individual mutants in both genes are female sterile under standard crossing conditions (light-dark cycles), an altered light regime enabled sexual development, which we found to be due to conditional female sterility of Δenv1, but not Δvel1. Phenotypes of growth and asexual sporulation as well as regulation of the peptide pheromone precursors of double mutants suggested that ENV1 and VEL1 balance positive and negative regulators of these functions. Additionally, VEL1 contributed to the strong deregulation of the pheromone system observed in env1 mutants. Female sterility of Δvel1 was rescued by deletion of env1 in darkness in MAT1-1, indicating a block of sexual development by ENV1 in darkness that is balanced by VEL1 in the wild-type. We conclude that ENV1 and VEL1 exert complementing functions in development of T. reesei. Our results further showed that the different developmental phenotypes of vel1/veA mutants in T. reesei and Aspergillus nidulans are not due to the presence or function of ENV1 in the VELVET regulatory pathway in T. reesei.

  16. Putative role of Tat-Env interaction in HIV infection.

    PubMed

    Poon, Selina; Moscoso, Carlos G; Xing, Li; Kan, Elaine; Sun, Yide; Kolatkar, Prasanna R; Vahlne, Anders G; Srivastava, Indresh K; Barnett, Susan W; Cheng, R Holland

    2013-09-24

    To study the complex formed between Tat protein and Env soluble trimeric immunogen, and compare with previously determined structures of Env native trimers and Env-CD4m complexes. The soluble Env trimer was used to mimic the spike glycoprotein on the virus surface for the study. To overcome limitations of other structural determination methods, cryoelectron microscopy was employed to image the complex, and single particle reconstruction was utilized to reconstruct the structure of the complex from collected micrographs. Molecular modeling of gp120-Tat was performed to provide atomic coordinates for docking. Images were preprocessed by multivariate statistical analysis to identify principal components of variation then submitted for reconstruction. Reconstructed structures were docked with modeled gp120-Tat atomic coordinates to study the positions of crucial epitopes. Analysis of the Env-Tat complex demonstrated an intermediate structure between Env native trimers and Env-CD4m structures. Docking results indicate that the CD4-binding site and the V3 loop are exposed in the Env-Tat complex. The integrin-binding sequence in Tat was also exposed in Env-Tat docking. The intermediate structure induced by Tat-interaction with Env could potentially provide an explanation for increased virus infection in the presence of Tat protein. Consequently, exposure of CD4-binding sites and a putative integrin-binding sequence on Tat in the complex may provide a new avenue for rational design of an effective HIV vaccine. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins

  17. Assembly and immunogenicity of chimeric Gag-Env proteins derived from the human immunodeficiency virus type 1.

    PubMed

    Truong, C; Brand, D; Mallet, F; Roingeard, P; Brunet, S; Barin, F

    1996-03-01

    We evaluated the potential of the precursor Gag protein (Pr55) of the human immunodeficiency virus type 1 (HIV-1) as a carrier for the presentation of envelope epitopes. Recombinant chimeric core-envelope protein-expressing constructs were derived by deletion of regions within the gag gene, especially of regions encoding p24 capsid epitopes. Sequences encoding either the principal neutralization determinant (PND) and/or the CD4-binding domains (CD4BS) were then inserted. Deletion of residues 196-226 within the p24 capsid protein did not prevent self-assembly into virus-like particles (VLPs) whereas deletion of residues 299-328 completely abolished VLP formation. Thus the major homology region (MHR) and proximal sequences are required for capsid assembly. An immunization study in mice showed that assembled chimeric proteins elicited strong anti-Gag, weak anti-envelope, and no neutralizing humoral responses. Nonassembled chimeric proteins were poor immunogens. Mapping of Pr55 antigenic sites using sera from immunized mice and peptides overlapping the entire Gag precursor showed that p24 capsid and p17 matrix epitopes presented to the immune system differed from the mature form (p24 or p17) and the multimeric immature form (Pr55).

  18. Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.

    PubMed Central

    Sodora, D L; Shpaer, E G; Kitchell, B E; Dow, S W; Hoover, E A; Mullins, J I

    1994-01-01

    Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered

  19. env-encoded residues are not required for transformation by p48v-myb.

    PubMed Central

    Lipsick, J S; Ibanez, C E

    1987-01-01

    The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus. Images PMID:3027417

  20. The Intracytoplasmic Domain of the Env Transmembrane Protein Is a Locus for Attenuation of Simian Immunodeficiency Virus SIVmac in Rhesus Macaques

    PubMed Central

    Shacklett, Barbara L.; Weber, Claudia Jo; Shaw, Karen E. S.; Keddie, Elise M.; Gardner, Murray B.; Sonigo, Pierre; Luciw, Paul A.

    2000-01-01

    The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques. PMID:10846063

  1. Study of the HIV-2 Env Cytoplasmic Tail Variability and Its Impact on Tat, Rev and Nef

    PubMed Central

    Bakouche, Nordine; Vandenbroucke, Anne-Thérèse; Goubau, Patrick; Ruelle, Jean

    2013-01-01

    Background The HIV-2 env’s 3’ end encodes the cytoplasmic tail (CT) of the Env protein. This genomic region also encodes the rev, Tat and Nef protein in overlapping reading frames. We studied the variability in the CT coding region in 46 clinical specimens and in 2 reference strains by sequencing and by culturing. The aims were to analyse the variability of Env CT and the evolution of proteins expressed from overlapping coding sequences. Results A 70% reduction of the length of the CT region affected the HIV-2 ROD and EHO strains in vitro due to a premature stop codon in the env gene. In clinical samples this wasn’t observed, but the CT length varied due to insertions and deletions. We noted 3 conserved and 3 variable regions in the CT. The conserved regions were those containing residues involved in Env endocytosis, the potential HIV-2 CT region implicated in the NF-kB activation and the potential end of the lentiviral lytic peptide one. The variable regions were the potential HIV-2 Kennedy region, the potential lentiviral lytic peptide two and the beginning of the potential lentiviral lytic peptide one. A very hydrophobic region was coded downstream of the premature stop codon observed in vitro, suggesting a membrane spanning region. Interestingly, the nucleotides that are responsible for the variability of the CT don’t impact rev and Nef. However, in the Kennedy-like coding region variability resulted only from nucleotide changes that impacted Env and Tat together. Conclusion The HIV-2 Env, Tat and Rev C-terminal part are subject to major length variations in both clinical samples and cultured strains. The HIV-2 Env CT contains variable and conserved regions. These regions don’t affect the rev and Nef amino acids composition which evolves independently. In contrast, Tat co-evolves with the Env CT. PMID:24223892

  2. Presence of env-like sequences in Quercus suber retrotransposons.

    PubMed

    Carvalho, M; Ribeiro, T; Viegas, W; Morais-Cecilio, L; Rocheta, M

    2010-01-01

    The main difference between LTR retrotransposons and retroviruses is the presence of the envelope (env) gene in the latter, downstream of the pol gene. The env gene is involved in their infectious capacity. Here we report the presence of env-like sequences in the genome of Quercus suber (cork oak), one of the most economically important Portuguese species. These gene sequences were isolated through DNA amplification between RNaseH conserved motifs and 3' LTR, based on the structure of copia retrotransposons. Phylogenetic analysis revealed that almost all the clones isolated are clustered with Cyclops-2, a Ty3-gypsy element identified in Pisum sativum, except one clustered with gypsy and copia retroelements found in different species. This suggests the existence of a potential ancestral sequence of the env gene, prior to the separation of Ty3-gypsy and Ty1-copia retrotransposons. Additionally, the isolated env-like sequences showed 26-39% of homology with env-like sequences characterized in viruses. The origin of env-like sequences in retrotransposons from host plant taxa is discussed.

  3. Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins

    PubMed Central

    Go, Eden P.; Cupo, Albert; Ringe, Rajesh; Pugach, Pavel; Moore, John P.

    2015-01-01

    ABSTRACT We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds. IMPORTANCE It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env

  4. Antagonism of BST-2/Tetherin Is a Conserved Function of the Env Glycoprotein of Primary HIV-2 Isolates.

    PubMed

    Chen, Chia-Yen; Shingai, Masashi; Welbourn, Sarah; Martin, Malcolm A; Borrego, Pedro; Taveira, Nuno; Strebel, Klaus

    2016-12-15

    Although HIV-2 does not encode a vpu gene, the ability to antagonize bone marrow stromal antigen 2 (BST-2) is conserved in some HIV-2 isolates, where it is controlled by the Env glycoprotein. We previously reported that a single-amino-acid difference between the laboratory-adapted ROD10 and ROD14 Envs controlled the enhancement of virus release (referred to here as Vpu-like) activity. Here, we investigated how conserved the Vpu-like activity is in primary HIV-2 isolates. We found that half of the 34 tested primary HIV-2 Env isolates obtained from 7 different patients enhanced virus release. Interestingly, most HIV-2 patients harbored a mixed population of viruses containing or lacking Vpu-like activity. Vpu-like activity and Envelope functionality varied significantly among Env isolates; however, there was no direct correlation between these two functions, suggesting they evolved independently. In comparing the Env sequences from one HIV-2 patient, we found that similar to the ROD10/ROD14 Envs, a single-amino-acid change (T568I) in the ectodomain of the TM subunit was sufficient to confer Vpu-like activity to an inactive Env variant. Surprisingly, however, absence of Vpu-like activity was not correlated with absence of BST-2 interaction. Taken together, our data suggest that maintaining the ability to antagonize BST-2 is of functional relevance not only to HIV-1 but also to HIV-2 as well. Our data show that as with Vpu, binding of HIV-2 Env to BST-2 is important but not sufficient for antagonism. Finally, as observed previously, the Vpu-like activity in HIV-2 Env can be controlled by single-residue changes in the TM subunit. Lentiviruses such as HIV-1 and HIV-2 encode accessory proteins whose function is to overcome host restriction mechanisms. Vpu is a well-studied HIV-1 accessory protein that enhances virus release by antagonizing the host restriction factor BST-2. HIV-2 does not encode a vpu gene. Instead, the HIV-2 Env glycoprotein was found to antagonize BST-2

  5. Prevalence of fowl glioma-inducing virus in chickens of zoological gardens in Japan and nucleotide variation in the env gene.

    PubMed

    Hatai, Hitoshi; Ochiai, Kenji; Murakami, Mariko; Imanishi, Syunsuke; Tomioka, Yukiko; Toyoda, Takeshi; Ohashi, Kazuhiko; Umemura, Takashi

    2008-05-01

    Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), is tumorigenic in the nervous system. In a zoological garden in Japan, approximately 40% of chickens, including Japanese fowls, were infected with FGV. Because this zoological garden plays a role as a major supplier of Japanese fowl for other zoological gardens, FGV infection is suspected to have spread among ornamental chickens. In this study, the prevalence of the disease was examined in a total of 129 chickens in three other zoological gardens by nested polymerase chain reaction (PCR), reverse transcription nested PCR and enzyme-linked immunosorbent assay. Twenty-six to 56 percent of the fowls in each of the examined gardens were positive by nested PCR. The phylogenetic analysis revealed that the 3' untranslated region, including the specific sequence of FGV, of the 14 isolated ALVs showed high sequence identity and a close relationship with FGV. In addition, the env gene of the isolates frequently showed mutations and deletions of nucleotides. These results suggest that FGV is prevalent among ornamental chickens kept in zoological gardens in Japan.

  6. Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.

    PubMed

    Han, Xiue; Zhang, Ping; Yu, Wei; Xiang, Wenhua; Li, Xiaodong

    2016-12-01

    The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAVL) in donkeys (EIAVD) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAVL) with attenuated strains serially passaged in donkey MDM (EIAVDLV) and donkey dermal cells (EIAVFDDV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAVFDDV infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAVFDDV. Results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum neutralizing antibodies that were also able to recognize parental viruses. This study indicates that these mutations played an important role in the attenuation of the EIAV vaccine strains.

  7. Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers.

    PubMed

    Go, Eden P; Ding, Haitao; Zhang, Shijian; Ringe, Rajesh P; Nicely, Nathan; Hua, David; Steinbock, Robert T; Golabek, Michael; Alin, James; Alam, S Munir; Cupo, Albert; Haynes, Barton F; Kappes, John C; Moore, John P; Sodroski, Joseph G; Desaire, Heather

    2017-05-01

    HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important

  8. Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

    PubMed Central

    Setz, Christian; Friedrich, Melanie; Schlößer, Stefan; Kölle, Julia; Spranger, Robert; Rauch, Pia; Reif, Tatjana; Karius-Fischer, Julia; Balasubramanyam, Ashok; Henklein, Petra; Fossen, Torgils; Schubert, Ulrich

    2017-01-01

    There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1. PMID:28388673

  9. A Comparative Phase I Study of Combination, Homologous Subtype-C DNA, MVA, and Env gp140 Protein/Adjuvant HIV Vaccines in Two Immunization Regimes

    PubMed Central

    Joseph, Sarah; Quinn, Killian; Greenwood, Aldona; Cope, Alethea V.; McKay, Paul F.; Hayes, Peter J.; Kopycinski, Jakub T.; Gilmour, Jill; Miller, Aleisha N.; Geldmacher, Christof; Nadai, Yuka; Ahmed, Mohamed I. M.; Montefiori, David C.; Dally, Len; Bouliotis, George; Lewis, David J. M.; Tatoud, Roger; Wagner, Ralf; Esteban, Mariano; Shattock, Robin J.; McCormack, Sheena; Weber, Jonathan

    2017-01-01

    There remains an urgent need for a prophylactic HIV vaccine. We compared combined MVA and adjuvanted gp140 to sequential MVA/gp140 after DNA priming. We expected Env-specific CD4+ T-cells after DNA and MVA priming, and Env-binding antibodies in 100% individuals after boosting with gp140 and that combined vaccines would not compromise safety and might augment immunogenicity. Forty volunteers were primed three times with DNA plasmids encoding (CN54) env and (ZM96) gag-pol-nef at 0, 4 and 8 weeks then boosted with MVA-C (CN54 env and gag-pol-nef) and glucopyranosyl lipid adjuvant—aqueous formulation (GLA-AF) adjuvanted CN54gp140. They were randomised to receive them in combination at the same visit at 16 and 20 weeks (accelerated) or sequentially with MVA-C at 16, 20, and GLA-AF/gp140 at 24 and 28 weeks (standard). All vaccinations were intramuscular. Primary outcomes included ≥grade 3 safety events and the titer of CN54gp140-specific binding IgG. Other outcomes included neutralization, binding antibody specificity and T-cell responses. Two participants experienced asymptomatic ≥grade 3 transaminitis leading to discontinuation of vaccinations, and three had grade 3 solicited local or systemic reactions. A total of 100% made anti-CN54gp140 IgG and combining vaccines did not significantly alter the response; geometric mean titer 6424 (accelerated) and 6578 (standard); neutralization of MW965.2 Tier 1 pseudovirus was superior in the standard group (82 versus 45% responders, p = 0.04). T-cell ELISpot responses were CD4+ and Env-dominant; 85 and 82% responding in the accelerated and standard groups, respectively. Vaccine-induced IgG responses targeted multiple regions within gp120 with the V3 region most immunodominant and no differences between groups detected. Combining MVA and gp140 vaccines did not result in increased adverse events and did not significantly impact upon the titer of Env-specific binding antibodies, which were seen in 100% individuals

  10. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  11. Interleukin-1- and Type I Interferon-Dependent Enhanced Immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef Vaccine Vector with Dual Deletions of Type I and Type II Interferon-Binding Proteins

    PubMed Central

    Delaloye, Julie; Filali-Mouhim, Abdelali; Cameron, Mark J.; Haddad, Elias K.; Harari, Alexandre; Goulet, Jean-Pierre; Gomez, Carmen E.; Perdiguero, Beatriz; Esteban, Mariano; Pantaleo, Giuseppe; Roger, Thierry; Sékaly, Rafick-Pierre

    2015-01-01

    ABSTRACT NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4+ T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV

  12. Interleukin-1- and type I interferon-dependent enhanced immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletions of type I and type II interferon-binding proteins.

    PubMed

    Delaloye, Julie; Filali-Mouhim, Abdelali; Cameron, Mark J; Haddad, Elias K; Harari, Alexandre; Goulet, Jean-Pierre; Gomez, Carmen E; Perdiguero, Beatriz; Esteban, Mariano; Pantaleo, Giuseppe; Roger, Thierry; Sékaly, Rafick-Pierre; Calandra, Thierry

    2015-04-01

    NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4(+) T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV

  13. Polymorphisms in the HIV-1 gp41 env gene, natural resistance to enfuvirtide (T-20) and pol resistance among pregnant Brazilian women.

    PubMed

    Reis, Mônica Nogueira da Guarda; de Alcântara, Keila Correa; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

    2014-01-01

    The selective pressure of antiretroviral drugs (ARVs) targeting HIV-1 pol can promote drug resistance mutations in other genomic regions, such as env. Drug resistance among women should be monitored to avoid horizontal and mother-to-child transmission. To describe natural resistance to T-20 (enfuvirtide), gp41 env polymorphisms, mutations in pol and HIV-1 subtypes, 124 pregnant women were recruited. For 98 patients, the gp41 env, protease (PR) and reverse transcriptase (RT) fragments were sequenced. The patients were ARV naïve (n = 30), taking mother-to-child transmission prophylaxis (n = 50), or being treated with highly active ARV therapy/HAART (n = 18). The Stanford and IAS/USA databases and other sources were used to analyze PR/RT, gp41 env resistance mutations. The HIV-1 genetic diversity was analyzed by REGA/phylogenetic analyses. The patients' median age was 25 years (range, 16-42), 18.4% had AIDS. The frequency of natural resistance to T-20 (N42D, L44M, and R46M-low-impact mutations) was 6.1% (6/98); 20.4% (20/98) had compensatory mutations in HR2. The prevalence of transmitted drug resistance in the pol was 13.3% (4/30), and the prevalence of secondary drug resistance was 33.3% (6/18). Two patients were infected with multidrug resistant/MDR viruses. The analysis of HIV-1 subtypes (PR/RT/gp41) revealed that 61.2% (60/98) were subtype B, 12.2% (12/98) were subtype C, 4.1% (4/98) were subtype F1, and 22.4% (22/98) were possible recombinants (BF1 = 20.4%; BC = 2%). Natural resistance to T-20 was not associated with pol resistance or previous ARV use. The high rate of secondary resistance, including MDR, indicates that the number of women that may need T-20 salvage therapy may be higher than anticipated.

  14. Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.

    PubMed

    Scharf, Louise; Wang, Haoqing; Gao, Han; Chen, Songye; McDowall, Alasdair W; Bjorkman, Pamela J

    2015-09-10

    The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.

  15. Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions.

    PubMed

    Zhou, Shuntai; Bednar, Maria M; Sturdevant, Christa B; Hauser, Blake M; Swanstrom, Ronald

    2016-08-15

    HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of ≤2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated. Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional

  16. ALV-J GP37 molecular analysis reveals novel virus-adapted sites and three tyrosine-based Env species.

    PubMed

    Ye, Jianqiang; Fan, Zhonglei; Shang, Jianjun; Tian, Xiaoyan; Yang, Jialiang; Chen, Hongjun; Shao, Hongxia; Qin, Aijian

    2015-01-01

    Compared to other avian leukosis viruses (ALV), ALV-J primarily induces myeloid leukemia and hemangioma and causes significant economic loss for the poultry industry. The ALV-J Env protein is hypothesized to be related to its unique pathogenesis. However, the molecular determinants of Env for ALV-J pathogenesis are unclear. In this study, we compared and analyzed GP37 of ALV-J Env and the EAV-HP sequence, which has high homology to that of ALV-J Env. Phylogenetic analysis revealed five groups of ALV-J GP37 and two novel ALV-J Envs with endemic GP85 and EAV-HP-like GP37. Furthermore, at least 15 virus-adapted mutations were detected in GP37 compared to the EAV-HP sequence. Further analysis demonstrated that three tyrosine-based motifs (YxxM, ITIM (immune tyrosine-based inhibitory motif) and ITAM-like (immune tyrosine-based active motif like)) associated with immune disease and oncogenesis were found in the cytoplasmic tail of GP37. Based on the potential function and distribution of these motifs in GP37, ALV-J Env was grouped into three species, inhibitory Env, bifunctional Env and active Env. Accordingly, 36.91%, 61.74% and 1.34% of ALV-J Env sequences from GenBank are classified as inhibitory, bifunctional and active Env, respectively. Additionally, the Env of the ALV-J prototype strain, HPRS-103, and 17 of 18 EAV-HP sequences belong to the inhibitory Env. And models for signal transduction of the three ALV-J Env species were predicted. Our findings and models provide novel insights for identifying the roles and molecular mechanism of ALV-J Env in the unique pathogenesis of ALV-J.

  17. Phylogenetic analysis of env, gag, and tat genes of HIV type 1 detected among the injecting drug users in West Bengal, India.

    PubMed

    Mullick, Ranajoy; Sengupta, Satarupa; Sarkar, Kamalesh; Saha, M K; Chakrabarti, Sekhar

    2006-12-01

    A recent occurrence of HIV-1 seropositivity among a group of injecting drug users (IDUs) in Darjeeling, a hilly district in northern West Bengal, revealed overall 11.8% HIV seroprevalence. Our study based on env (C2-V3), gag (p24-p7), and tat (exon-1) genomic regions of HIV-1 detected among this population showed that Darjeeling IDU sequences belonged to subtype C. Interestingly, the IDU sequences from Darjeeling were again found to be closer to the C strains from Manipur, a northeastern state in India, which is linked to the Golden Triangle via the Manipur-Myanmar border, rather than the IDU C sequences from Nepal, a neighboring country of India. The outgroup reference strains from different sites of IDU-driven epidemics in the world like Russia, Vietnam, Thailand, and Spain belonged to the nonsubtype C group and formed separate clusters from the subtype C cluster in our analysis. These results indicate a rapid spread of HIV-1 by possible drug trafficking along international boundaries, which might also help in the invasion of HIV-1 among IDUs of Darjeeling through the Manipur-Myanmar border of India.

  18. Targeting HIV-1 Env gp140 to LOX-1 Elicits Immune Responses in Rhesus Macaques.

    PubMed

    Zurawski, Gerard; Zurawski, Sandra; Flamar, Anne-Laure; Richert, Laura; Wagner, Ralf; Tomaras, Georgia D; Montefiori, David C; Roederer, Mario; Ferrari, Guido; Lacabaratz, Christine; Bonnabau, Henri; Klucar, Peter; Wang, Zhiqing; Foulds, Kathryn E; Kao, Shing-Fen; Yates, Nicole L; LaBranche, Celia; Jacobs, Bertram L; Kibler, Karen; Asbach, Benedikt; Kliche, Alexander; Salazar, Andres; Reed, Steve; Self, Steve; Gottardo, Raphael; Galmin, Lindsey; Weiss, Deborah; Cristillo, Anthony; Thiebaut, Rodolphe; Pantaleo, Giuseppe; Levy, Yves

    2016-01-01

    Improved antigenicity against HIV-1 envelope (Env) protein is needed to elicit vaccine-induced protective immunity in humans. Here we describe the first tests in non-human primates (NHPs) of Env gp140 protein fused to a humanized anti-LOX-1 recombinant antibody for delivering Env directly to LOX-1-bearing antigen presenting cells, especially dendritic cells (DC). LOX-1, or 1ectin-like oxidized low-density lipoprotein (LDL) receptor-1, is expressed on various antigen presenting cells and endothelial cells, and is involved in promoting humoral immune responses. The anti-LOX-1 Env gp140 fusion protein was tested for priming immune responses and boosting responses in animals primed with replication competent NYVAC-KC Env gp140 vaccinia virus. Anti-LOX-1 Env gp140 vaccination elicited robust cellular and humoral responses when used for either priming or boosting immunity. Co-administration with Poly ICLC, a TLR3 agonist, was superior to GLA, a TLR4 agonist. Both CD4+ and CD8+ Env-specific T cell responses were elicited by anti-LOX-1 Env gp140, but in particular the CD4+ T cells were multifunctional and directed to multiple epitopes. Serum IgG and IgA antibody responses induced by anti-LOX-1 Env gp140 against various gp140 domains were cross-reactive across HIV-1 clades; however, the sera neutralized only HIV-1 bearing sequences most similar to the clade C 96ZM651 Env gp140 carried by the anti-LOX-1 vehicle. These data, as well as the safety of this protein vaccine, justify further exploration of this DC-targeting vaccine approach for protective immunity against HIV-1.

  19. Targeting HIV-1 Env gp140 to LOX-1 Elicits Immune Responses in Rhesus Macaques

    PubMed Central

    Zurawski, Sandra; Flamar, Anne-Laure; Richert, Laura; Wagner, Ralf; Tomaras, Georgia D.; Montefiori, David C.; Roederer, Mario; Ferrari, Guido; Lacabaratz, Christine; Bonnabau, Henri; Klucar, Peter; Wang, Zhiqing; Foulds, Kathryn E.; Kao, Shing-Fen; Yates, Nicole L.; LaBranche, Celia; Jacobs, Bertram L.; Kibler, Karen; Asbach, Benedikt; Kliche, Alexander; Salazar, Andres; Reed, Steve; Self, Steve; Gottardo, Raphael; Galmin, Lindsey; Weiss, Deborah; Cristillo, Anthony; Thiebaut, Rodolphe; Pantaleo, Giuseppe; Levy, Yves

    2016-01-01

    Improved antigenicity against HIV-1 envelope (Env) protein is needed to elicit vaccine-induced protective immunity in humans. Here we describe the first tests in non-human primates (NHPs) of Env gp140 protein fused to a humanized anti-LOX-1 recombinant antibody for delivering Env directly to LOX-1-bearing antigen presenting cells, especially dendritic cells (DC). LOX-1, or 1ectin-like oxidized low-density lipoprotein (LDL) receptor-1, is expressed on various antigen presenting cells and endothelial cells, and is involved in promoting humoral immune responses. The anti-LOX-1 Env gp140 fusion protein was tested for priming immune responses and boosting responses in animals primed with replication competent NYVAC-KC Env gp140 vaccinia virus. Anti-LOX-1 Env gp140 vaccination elicited robust cellular and humoral responses when used for either priming or boosting immunity. Co-administration with Poly ICLC, a TLR3 agonist, was superior to GLA, a TLR4 agonist. Both CD4+ and CD8+ Env-specific T cell responses were elicited by anti-LOX-1 Env gp140, but in particular the CD4+ T cells were multifunctional and directed to multiple epitopes. Serum IgG and IgA antibody responses induced by anti-LOX-1 Env gp140 against various gp140 domains were cross-reactive across HIV-1 clades; however, the sera neutralized only HIV-1 bearing sequences most similar to the clade C 96ZM651 Env gp140 carried by the anti-LOX-1 vehicle. These data, as well as the safety of this protein vaccine, justify further exploration of this DC-targeting vaccine approach for protective immunity against HIV-1. PMID:27077384

  20. Cocirculation of Two env Molecular Variants, of Possible Recombinant Origin, in Gorilla and Chimpanzee Simian Foamy Virus Strains from Central Africa

    PubMed Central

    Richard, Léa; Rua, Réjane; Betsem, Edouard; Mouinga-Ondémé, Augustin; Kazanji, Mirdad; Leroy, Eric; Njouom, Richard; Buseyne, Florence; Afonso, Philippe V.

    2015-01-01

    ABSTRACT Simian foamy virus (SFV) is a ubiquitous retrovirus in nonhuman primates (NHPs) that can be transmitted to humans, mostly through severe bites. In the past few years, our laboratory has identified more than 50 hunters from central Africa infected with zoonotic SFVs. Analysis of the complete sequences of five SFVs obtained from these individuals revealed that env was the most variable gene. Furthermore, recombinant SFV strains, some of which involve sequences in the env gene, were recently identified. Here, we investigated the variability of the env genes of zoonotic SFV strains and searched for possible recombinants. We sequenced the complete env gene or its surface glycoprotein region (SU) from DNA amplified from the blood of (i) a series of 40 individuals from Cameroon or Gabon infected with a gorilla or chimpanzee foamy virus (FV) strain and (ii) 1 gorilla and 3 infected chimpanzees living in the same areas as these hunters. Phylogenetic analyses revealed the existence of two env variants among both the gorilla and chimpanzee FV strains that were present in zoonotic and NHP strains. These variants differ greatly (>30% variability) in a 753-bp-long region located in the receptor-binding domain of SU, whereas the rest of the gene is very conserved. Although the organizations of the Env protein sequences are similar, the potential glycosylation patterns differ between variants. Analysis of recombination suggests that the variants emerged through recombination between different strains, although all parental strains could not be identified. IMPORTANCE SFV infection in humans is a great example of a zoonotic retroviral infection that has not spread among human populations, in contrast to human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). Recombination was a major mechanism leading to the emergence of HIV. Here, we show that two SFV molecular envelope gene variants circulate among ape populations in Central Africa and that both

  1. Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline.

    PubMed

    Chalvet, F; Teysset, L; Terzian, C; Prud'homme, N; Santamaria, P; Bucheton, A; Pélisson, A

    1999-05-04

    Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.

  2. Isolation and sequencing of infectious clones of feline foamy virus and a human/feline foamy virus Env chimera.

    PubMed

    Hatama, S; Otake, K; Omoto, S; Murase, Y; Ikemoto, A; Mochizuki, M; Takahashi, E; Okuyama, H; Fujii, Y

    2001-12-01

    Full-length DNAs of the Coleman and S7801 strains (pSKY3.0, pSKY5.0) of infectious feline foamy viruses (FFVs) were cloned and sequenced. Parental viruses, designated SKY3.0 and SKY5.0, were secreted following transfection of Crandell feline kidney (CRFK) cells. Production of the rescued parental viruses was enhanced in the presence of trichostatin A. Amino acid sequence similarities between FFV and human foamy virus (HFV) are extremely low for the envelope protein and capsid antigen, as predicted from the two clones. However, a chimeric FFV clone was constructed with the HFV Env substituted for the FFV Env. The chimeric virus (HFFV, SKY4.0) was able to infect and replicate in CRFK cells as well as in peripheral blood mononuclear cells of cats in vivo. Consequently, the chimeric HFFV may be useful for the creation of FV vectors for gene transfer strategies.

  3. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic.

    PubMed

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-10-11

    Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame -2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins-an expected outcome with overlaps in frame -2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic.

  4. Pseudotyping incompatibility between HIV-1 and gibbon ape leukemia virus Env is modulated by Vpu.

    PubMed

    Lucas, Tiffany M; Lyddon, Terri D; Cannon, Paula M; Johnson, Marc C

    2010-03-01

    The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu(RD), was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.

  5. Functional and structural characterization of EnvZ, an osmosensing histidine kinase of E. coli.

    PubMed

    Yoshida, Takeshi; Phadtare, Sangita; Inouye, Masayori

    2007-01-01

    EnvZ is an osmosensing histidine kinase located in the inner membrane, and one of the most extensively studied Escherichia coli histidine kinases. Because of its structural complexity, functional and structural studies have been quite challenging. It is a multidomain transmembrane protein consisting of 450 amino acid residues. In addition, it must form a dimer to function as a histidine kinase like all the other histidine kinases. EnvZ consists of the 115-residue periplasmic domain, two transmembrane domains (TM1 and TM2), and the cytoplasmic domain consisting of the 43-residue linker (HAMP) domain and the 228-residue kinase domain. It has been shown that the kinase domain of EnvZ, responsible for its enzymatic activities, contains all of the conserved regions of histidine kinases such as H, F, N, G1, G2, and G3 boxes. Therefore, the 271-residue cytoplasmic domain of EnvZ (termed EnvZc) has been used as a model system to establish fundamental characteristics of histidine kinases. The DNA fragment encoding EnvZc was cloned in pET vector and EnvZc was expressed and purified. It is highly soluble and retains all the enzymatic activities of EnvZ. We demonstrated that it consists of two functional domains, domain A and domain B. NMR spectroscopic studies of these two domains revealed, for the first time, the structure of a histidine kinase. Domain A is responsible for dimerization of EnvZc forming a four-helical bundle containing two alpha-helical hairpin structures, while domain B is a monomer and has an ATP-binding pocket formed by regions conserved among the histidine kinases. In this chapter, we describe functional and structural studies of EnvZc, which can be applied to characterize other histidine kinases.

  6. Modulation of inv gene expression by the OmpR two-component response regulator protein of Yersinia enterocolitica.

    PubMed

    Raczkowska, A; Brzóstkowska, M; Kwiatek, A; Bielecki, J; Brzostek, K

    2011-07-01

    To elucidate the physiological meaning of OmpR-dependent expression of invasin gene (inv) inhibition in Yersinia enterocolitica, the function of the EnvZ/OmpR regulatory pathway in osmoregulation of inv expression was analyzed in detail. The osmoregulation of inv expression was found to be a multifaceted process involving both OmpR-dependent and -independent mechanisms. Analysis of inv transcription in strains lacking OmpR or EnvZ proteins indicated that kinase EnvZ is not the only regulator of OmpR phosphorylation. Using the transcriptional inv::lacZ fusion in a heterologous system (Escherichia coli) we tried to clarify the role of OmpR in the inv regulatory circuit composed of negative (H-NS) and positive (RovA) regulators of inv gene transcription. We were able to show a significant increase in inv expression in E. coli ompR background under H-NS( Ecoli )-repressed condition. Moreover, H-NS-mediated inv repression was relieved when RovA of Y. enterocolitica was expressed from a plasmid. Furthermore, we showed that RovA may activate inv expression irrespective on the presence of H-NS protein. Using this strategy we showed that OmpR of Y. enterocolitica decrease RovA-mediated inv activation.

  7. Downregulation of Human Endogenous Retrovirus Type K (HERV-K) Viral env RNA in Pancreatic Cancer Cells Decreases Cell Proliferation and Tumor Growth.

    PubMed

    Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z; Li, Donghui; Johanning, Gary L; Wang-Johanning, Feng

    2017-10-01

    Purpose: We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer.Experimental Design: shRNA was employed to knockdown (KD) the expression of HERV-K in pancreatic cancer cells.Results: HERV-K env expression was detected in seven pancreatic cancer cell lines and in 80% of pancreatic cancer patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several pancreatic cancer cell lines. Reverse transcriptase activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in pancreatic cancer patient sera (N = 106) than in normal donor sera (N = 40). Importantly, the in vitro and in vivo growth rates of three pancreatic cancer cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-Seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several pancreatic cancer cells or tumors.Conclusions: These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in pancreatic cancer. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of pancreatic cancer, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis, and immunotherapy of pancreatic cancer. Clin Cancer Res; 23(19); 5892-911. ©2017 AACR. ©2017 American Association for Cancer Research.

  8. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic

    PubMed Central

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-01-01

    Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame −2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp. Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins—an expected outcome with overlaps in frame −2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic. PMID:27681623

  9. Quantifying Selection against Synonymous Mutations in HIV-1 env Evolution

    PubMed Central

    Zanini, Fabio

    2013-01-01

    Intrapatient evolution of human immunodeficiency virus type 1 (HIV-1) is driven by the adaptive immune system resulting in rapid change of HIV-1 proteins. When cytotoxic CD8+ T cells or neutralizing antibodies target a new epitope, the virus often escapes via nonsynonymous mutations that impair recognition. Synonymous mutations do not affect this interplay and are often assumed to be neutral. We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. We find that most synonymous variants are lost even though they often reach high frequencies in the viral population, suggesting a cost to the virus. Using published data from SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) assays, we find that synonymous mutations that disrupt base pairs in RNA stems flanking the variable loops of gp120 are more likely to be lost than other synonymous changes: these RNA hairpins might be important for HIV-1. Computational modeling indicates that, to be consistent with the data, a large fraction of synonymous mutations in this genomic region need to be deleterious with a cost on the order of 0.002 per day. This weak selection against synonymous substitutions does not result in a strong pattern of conservation in cross-sectional data but slows down the rate of evolution considerably. Our findings are consistent with the notion that large-scale patterns of RNA structure are functionally relevant, whereas the precise base pairing pattern is not. PMID:23986591

  10. Construction of infectious feline foamy virus genomes: cat antisera do not cross-neutralize feline foamy virus chimera with serotype-specific Env sequences.

    PubMed

    Zemba, M; Alke, A; Bodem, J; Winkler, I G; Flower, R L; Pfrepper, K; Delius, H; Flügel, R M; Löchelt, M

    2000-01-05

    Full-length genomes of the feline foamy virus (FFV or FeFV) isolate FUV were constructed. DNA clone pFeFV-7 stably directed the expression of infectious FFV progeny virus indistinguishable from wild-type, uncloned FFV isolate FUV. The env and bel 1 genes of pFeFV-7 were substituted for by corresponding sequences of the FFV serotype 951 since previous studies implicated a defined part of FFV Env protein as responsible for serotype-specific differences in serum neutralization (I. G. Winkler, R. M. Flügel, M. Löchelt, and R. L. P. Flower, 1998. Virology 247: 144-151). Recombinant virus derived from chimeric plasmid pFeFV-7/951 containing the hybrid env gene and the parental clone pFeFV-7 were used for neutralization studies. By means of a rapid titration assay for FFV infectivity, we show that progeny virus derived from plasmid pFeFV-7 was neutralized by FUV- but not by 951-specific antisera, whereas pFeFV-7/951-derived chimeric virus was neutralized by 951-specific antisera only. Both recombinant proviruses will be useful for repeated delivery of foreign genes for therapeutic gene applications into cats.

  11. Sequencing of Gag/Env association with HIV genotyping resolution and HIV-related epidemiologic studies of HIV in China.

    PubMed

    Ren, L; Wang, H W; Xu, Y; Feng, Y; Zhang, H F; Wang, K H

    2016-10-24

    HIV genotyping has led to conflicting results between laboratories. Therefore, identifying the most accurate gene combinations to sequence remains a priority. Datasets of Chinese HIV subtypes based on several markers and deposited in PubMed, Metstr, CNKI, and VIP databases between 2000 and 2015 were studied. In total, 9177 cases of amplification-positive samples from 26 provinces of China were collected and used to classify HIV subtypes based on eight individual genes or a combination thereof. CRF01_AE, CRF07_BC, CRF08_BC and B were the prevalent HIV subtypes in China, accounting for 84.07% of all genotypes. Gag/Env sequencing classified a greater number of HIV subtypes compared to other genes or combination of gene fragments. The geographical distribution of Gag and Gag/Env genotypes was similar to that observed with all genetic markers. Further principal component analysis showed a significantly different geographical distribution pattern of HIV in China for HIV genotypes detected with Gag/Env, which was in line with the distribution of all HIV genotypes in China. Gag/Env sequences had the highest diversity of the eight markers studied, followed by Gag and Gag/Pol/Env; Pol/Env polymorphisms were the least divergent. Gag/Env can serve as a high-resolution marker for HIV genotyping.

  12. v-mos proteins encoded by myeloproliferative sarcoma virus and its ts159 mutant.

    PubMed Central

    Singh, B; Stocking, C; Walker, R; Yang, Y D; Ostertag, W; Arlinghaus, R B

    1992-01-01

    The myeloproliferative sarcoma virus (MPSV) v-mos protein was predicted to be identical in size to p39c-mos because of an observed one-base deletion in the seventh codon of the env-mos open reading frame, which would allow translation to initiate at the methionine equivalent to codon 32 of the env-mos gene. On the basis of published results, p39c-mos is known to have greatly reduced in vitro protein kinase activity compared with p37env-mos encoded by Moloney murine sarcoma virus. Unexpectedly, the relative activity of the MPSV v-mos protein kinase was comparable to that of p37env-mos. Consistent with this finding, the size of MPSV v-mos protein was found to be similar to the size of p37env-mos. Moreover, the pattern and sizes of phosphorylated bands produced by autophosphorylation of the MPSV v-mos protein were similar to those of p37env-mos. These results were confirmed by in vitro transcription-translation of the MPSV v-mos gene. Resequencing portions of the MPSV mos gene failed to show the deletion within codon 7. Except for the codon 262 deletion, other mutations characteristic of MPSV and temperature-sensitive MPSV v-mos genes were confirmed. A glycine-to-arginine mutation at residue 338 of the MPSV env-mos sequence, previously shown to cause thermosensitivity of the mutant virus (termed ts159) transforming function, yielded a v-mos protein that had significantly reduced protein kinase activity in vitro. These findings indicate that MPSV, like other Moloney murine sarcoma virus strains, also encodes a functional env-mos protein. Images PMID:1309903

  13. Cross-Neutralizing Antibodies in HIV-1 Individuals Infected by Subtypes B, F1, C or the B/Bbr Variant in Relation to the Genetics and Biochemical Characteristics of the env Gene

    PubMed Central

    de Almeida, Dalziza Victalina; Macieira, Karine Venegas; Grinsztejn, Beatriz Gilda Jegerhorn; Veloso dos Santos, Valdiléa Gonçalves; Guimarães, Monick Lindenmeyer

    2016-01-01

    Various HIV-1 env genetic and biochemical features impact the elicitation of cross-reactive neutralizing antibodies in natural infections. Thus, we aimed to investigate cross-neutralizing antibodies in individuals infected with HIV-1 env subtypes B, F1, C or the B/Bbr variant as well as env characteristics. Therefore, plasma samples from Brazilian chronically HIV-1 infected individuals were submitted to the TZM-bl neutralization assay. We also analyzed putative N-glycosylation sites (PNGLs) and the size of gp120 variable domains in the context of HIV-1 subtypes prevalent in Brazil. We observed a greater breadth and potency of the anti-Env neutralizing response in individuals infected with the F1 or B HIV-1 subtypes compared with the C subtype and the variant B/Bbr. We observed greater V1 B/Bbr and smaller V4 F1 than those of other subtypes (p<0.005), however neither was there a correlation verified between the variable region length and neutralization potency, nor between PNLG and HIV-1 subtypes. The enrichment of W at top of V3 loop in weak neutralizing response viruses and the P in viruses with higher neutralization susceptibility was statistically significant (p = 0.013). Some other signatures sites were associated to HIV-1 subtype-specific F1 and B/Bbr samples might influence in the distinct neutralizing response. These results indicate that a single amino acid substitution may lead to a distinct conformational exposure or load in the association domain of the trimer of gp120 and interfere with the induction power of the neutralizing response, which affects the sensitivity of the neutralizing antibody and has significant implications for vaccine design. PMID:27936047

  14. Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii)

    USGS Publications Warehouse

    Emmenegger, Eveline J.; Glenn, Jolene A.; Winton, James R.; Batts, William N.; Gregg, Jacob L.; Hershberger, Paul K.

    2014-01-01

    Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

  15. Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).

    PubMed

    Emmenegger, Eveline J; Glenn, Jolene A; Winton, James R; Batts, William N; Gregg, Jacob L; Hershberger, Paul K

    2014-11-07

    Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

  16. Protein polymer: Gene libraries open up

    NASA Astrophysics Data System (ADS)

    Ding, Sheng; Wang, Xiaoxiao; Barron, Annelise E.

    2011-02-01

    By combining gene cloning and amplification techniques, a new one-pot, parallel synthesis method for the generation of long, repetitive genes is realized. The method promises to open up the discovery of protein polymer biomaterials.

  17. Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates

    PubMed Central

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan; Seaman, Michael; Montefiori, David C.; Labranche, Celia; Yates, Nicole L.; Shen, Xiaoying; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; McDermott, Adrian; Kao, Shing-Fen; Roederer, Mario; Hawkins, Natalie; Self, Steve; Yao, Jiansheng; Farrell, Patrick; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony; Weiss, Deborah; Lee, Carter; Kibler, Karen; Jacobs, Bert; Asbach, Benedikt; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe

    2015-01-01

    ABSTRACT We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4+ T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8+ T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that

  18. From Gene Mutation to Protein Characterization

    ERIC Educational Resources Information Center

    Moffet, David A.

    2009-01-01

    A seven-week "gene to protein" laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the…

  19. From Gene Mutation to Protein Characterization

    ERIC Educational Resources Information Center

    Moffet, David A.

    2009-01-01

    A seven-week "gene to protein" laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the…

  20. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    SciTech Connect

    Shi, Jian; Zhang, Huaidong; Gong, Rui; Xiao, Gengfu

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  2. The lumQ gene is linked to the lumP gene and the lux operon in Photobacterium leiognathi.

    PubMed

    Lin, J W; Yu, K Y; Chao, Y F; Weng, S F

    1995-12-14

    The nucleotide sequence of the designated lumQ gene (EMBL accession No. U35231) from Photobacterium leiognathi PL741 has been determined, and the encoded amino acid sequence is deduced. The LumQ protein has a calculated M(r) of 28,416 and comprises 248 amino acid residues. The lumQ gene is identified as the envY-like gene by significant similarity of the encoded protein with the EnvY and AdiY proteins of E. coli; there the envY gene encodes the porin thermoregulatory protein EnvY, and the adiY gene encodes the putative transcriptional regulator protein AdiY. It suggests that the lumQ gene of P. leiognathi is orthologous to the envY and adiY genes of E. coli. The function of the protein encoded by the lumQ gene from P. leiognathi is not really defined yet, it is likely to be the DNA-binding protein related to the araC and xylS family of transcriptional regulators. The lumQ and lumP genes form the lum operon which linked to the lux operon, but run in the opposite direction. The gene order of the lum and the lux operon is < -ter-lumQ-lumP-R&R-luxC-luxD-luxA-luxB- luxN-luxE- > (R&R: regulatory region; ter: transcriptional terminator); whereas the regulatory region (R&R) includes two promoter systems, PR-promoter for the lux operon and PL-promoter for the lum operon; ter is the transcriptional terminator of the lum operon.

  3. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  4. Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL.

    PubMed Central

    Carmichael, A; Jin, X; Sissons, P

    1996-01-01

    Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are

  5. Integrated protein function prediction by mining function associations, sequences, and protein-protein and gene-gene interaction networks.

    PubMed

    Cao, Renzhi; Cheng, Jianlin

    2016-01-15

    Protein function prediction is an important and challenging problem in bioinformatics and computational biology. Functionally relevant biological information such as protein sequences, gene expression, and protein-protein interactions has been used mostly separately for protein function prediction. One of the major challenges is how to effectively integrate multiple sources of both traditional and new information such as spatial gene-gene interaction networks generated from chromosomal conformation data together to improve protein function prediction. In this work, we developed three different probabilistic scores (MIS, SEQ, and NET score) to combine protein sequence, function associations, and protein-protein interaction and spatial gene-gene interaction networks for protein function prediction. The MIS score is mainly generated from homologous proteins found by PSI-BLAST search, and also association rules between Gene Ontology terms, which are learned by mining the Swiss-Prot database. The SEQ score is generated from protein sequences. The NET score is generated from protein-protein interaction and spatial gene-gene interaction networks. These three scores were combined in a new Statistical Multiple Integrative Scoring System (SMISS) to predict protein function. We tested SMISS on the data set of 2011 Critical Assessment of Function Annotation (CAFA). The method performed substantially better than three base-line methods and an advanced method based on protein profile-sequence comparison, profile-profile comparison, and domain co-occurrence networks according to the maximum F-measure. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  7. Real-time analysis of human immunodeficiency virus type 1 Env-mediated membrane fusion by fluorescence resonance energy transfer.

    PubMed

    Furuta, Rika A; Nishikawa, Masao; Fujisawa, Jun-ichi

    2006-02-01

    Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-mediated membrane fusion occurs as a sequence of events that is triggered by CD4 binding to the Env gp120 subunit. In this study, we analyzed the dynamics of Env-mediated membrane fusion at the single-cell level using fluorescent fusion proteins and confocal laser fluorescent microscopy. Either enhanced cyan or yellow fluorescent protein (CFP and YFP, respectively) was fused to the end of the cytoplasmic regions of the HIV-1 receptors (CD4 and CCR5) and Env proteins. Real-time imaging of membrane fusion mediated by these recombinant proteins revealed that the kinetics of fusion in our system was faster than that previously reported. Analysis of the receptor interaction by fluorescence resonance energy transfer (FRET) at the single-cell level demonstrated a tendency for oligomerization of CD4-CD4, but not of CD4-CCR5, in the absence of Env-expressing cells. However, when Env-expressing cells attached to the receptor cells, FRET produced by CD4-CCR5 interaction was increased; the FRET intensity began to decline before the formation of the fusion pore. These changes in FRET may represent the temporal association of these receptors, triggered by gp120 binding, and their dissociation during the formation of the fusion pore. In addition, the FRET analysis of receptor interactions in the presence of fusion inhibitors showed that not only inhibitors acting on CCR5 but also the gp41-derived peptide T-20 interfered with CD4-CCR5 interaction during fusion. These data suggest that T-20 could affect the formation of Env-receptors complexes during the membrane fusion.

  8. Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex

    PubMed Central

    Ochsenbauer-Jambor, Christina; Delos, Sue E.; Accavitti, Mary Ann; White, Judith M.; Hunter, Eric

    2002-01-01

    We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets

  9. Impact of antibody quality and anamnestic response on viremia control post-challenge in a combined Tat/Env vaccine regimen in rhesus macaques

    PubMed Central

    Demberg, Thorsten; Brocca-Cofano, Egidio; Kuate, Seraphin; Aladi, Stanley; Vargas-Inchaustegui, Diego A.; Venzon, David; Kalisz, Irene; Kalyanaraman, V.S.; Lee, Eun Mi; Pal, Ranajit; DiPasquale, Janet; Ruprecht, Ruth M.; Montefiori, David C.; Srivastava, Indresh; Barnett, Susan W.; Robert-Guroff, Marjorie

    2013-01-01

    Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy. PMID:23528732

  10. Dense display of HIV-1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to HIV-1 Env.

    PubMed

    Mattiacio, Jonelle; Walter, Scott; Brewer, Matt; Domm, William; Friedman, Alan E; Dewhurst, Stephen

    2011-03-21

    The generation of strong, virus-neutralizing antibody responses to the HIV-1 envelope spike (Env) is a major goal in HIV-1 vaccine research. To try to enhance the Env-specific response, we displayed oligomeric gp140 on a virus-like scaffold provided by the lambda phage capsid. To do this, an in vitro complementation system was used to "decorate" phage particles with glycosylated, mammalian cell-derived envelope oligomers. We compared the immune response to lambda phage particles displaying HIV-1 Env to that elicited by soluble oligomeric gp140 in rabbits. Env-binding antibody titers were higher in animals that received oligomeric gp140 as compared to Env decorated phage particles, as were virus neutralizing antibody responses. The Env decorated phage particles were, however, able to efficiently boost a protein-primed humoral response to levels equivalent to those elicited by high-dose adjuvanted Env oligomers. These results show that display of HIV-1 envelope spikes on the bacteriophage lambda capsid does not result in an improved, Env-specific humoral immune response. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. HIV-1 Nef responsiveness is determined by Env variable regions involved in trimer association and correlates with neutralization sensitivity.

    PubMed

    Usami, Yoshiko; Göttlinger, Heinrich

    2013-11-14

    HIV-1 Nef and the unrelated murine leukemia virus glycoGag similarly enhance the infectivity of HIV-1 virions. We now show that the effects of Nef and glycoGag are similarly determined by variable regions of HIV-1 gp120 that control Env trimer association and neutralization sensitivity. Whereas neutralization-sensitive X4-tropic Env proteins conferred high responsiveness to Nef and glycoGag, particles bearing neutralization-resistant R5-tropic Envs were considerably less affected. The profoundly different Nef/glycoGag responsiveness of a neutralization-resistant and a neutralization-sensitive R5-tropic Env could be switched by exchanging their gp120 V1/V2 regions, which also switches their neutralization sensitivity. Within V1/V2, the same determinants governed Nef/glycoGag responsiveness and neutralization sensitivity, indicating that these phenotypes are mechanistically linked. The V1/V2 and V3 regions, which form an apical trimer-association domain, together determined the Nef and glycoGag responsiveness of an X4-tropic Env. Our results suggest that Nef and glycoGag counteract the inactivation of Env spikes with relatively unstable apical trimer-association domains.

  12. Making the Chromosome-Gene-Protein Connection.

    ERIC Educational Resources Information Center

    Mulvihill, Charlotte

    1996-01-01

    Presents an exercise that demonstrates the chromosome-gene-protein connection using sickle-cell anemia, a genetic disease with a well-characterized molecular basis. Involves connecting changes in DNA to protein outcomes and tying them into the next generation by meiosis and gamete formation with genetic crosses. Motivates students to integrate…

  13. Making the Chromosome-Gene-Protein Connection.

    ERIC Educational Resources Information Center

    Mulvihill, Charlotte

    1996-01-01

    Presents an exercise that demonstrates the chromosome-gene-protein connection using sickle-cell anemia, a genetic disease with a well-characterized molecular basis. Involves connecting changes in DNA to protein outcomes and tying them into the next generation by meiosis and gamete formation with genetic crosses. Motivates students to integrate…

  14. Unique N-linked glycosylation of CasBrE Env influences its stability, processing, and viral infectivity but not its neurotoxicity.

    PubMed

    Renszel, Krystal M; Traister, Russell S; Lynch, William P

    2013-08-01

    The envelope protein (Env) from the CasBrE murine leukemia virus (MLV) can cause acute spongiform neurodegeneration analogous to that induced by prions. Upon central nervous system (CNS) infection, Env is expressed as multiple isoforms owing to differential asparagine (N)-linked glycosylation. Because N-glycosylation can affect protein folding, stability, and quality control, we explored whether unique CasBrE Env glycosylation features could influence neurovirulence. CasBrE Env possesses 6/8 consensus MLV glycosylation sites (gs) but is missing gs3 and gs5 and contains a putative site (gs*). Twenty-nine mutants were generated by modifying these three sites, individually or in combination, to mimic the amino acid sequence in the nonneurovirulent Friend 57 MLV. Three basic viral phenotypes were observed: replication defective (dead; titer < 1 focus-forming unit [FFU]/ml), replication compromised (RC) (titer = 10(2) to 10(5) FFU/ml); and wild-type-like (WTL) (titer > 10(5) FFU/ml). Env protein was undetectable in dead mutants, while RC and WTL mutants showed variations in Env expression, processing, virus incorporation, virus entry, and virus spread. The newly introduced gs3 and gs5 sites were glycosylated, whereas gs* was not. Six WTL mutants tested in mice showed no clear attenuation in disease onset or severity versus controls. Furthermore, three RC viruses tested by neural stem cell (NSC)-mediated brainstem dissemination also induced acute spongiosis. Thus, while unique N-glycosylation affected structural features of Env involved in protein stability, proteolytic processing, and virus assembly and entry, these changes had minimal impact on CasBrE Env neurotoxicity. These findings suggest that the Env protein domains responsible for spongiogenesis represent highly stable elements upon which the more variable viral functional domains have evolved.

  15. Mucosal immunization of sheep with a Maedi-Visna virus (MVV) env DNA vaccine protects against early MVV productive infection.

    PubMed

    González, Belén; Reina, Ramsés; García, Iker; Andrés, Sara; Glaria, Idoia; Alzueta, María; Mora, María Isabel; Jugo, Begoña M; Arrieta-Aguirre, Inés; de la Lastra, José M Pérez; Rodríguez, Dolores; Rodríguez, Juan Ramón; Esteban, Mariano; Grilló, María Jesús; Blacklaws, Barbara A; Harkiss, Gordon D; Chebloune, Yahia; Luján, Lluís; de Andrés, Damián; Amorena, Beatriz

    2005-07-29

    Gene gun mucosal DNA immunization of sheep with a plasmid expressing the env gene of Maedi-Visna virus (MVV) was used to examine the protection against MVV infection in sheep from a naturally infected flock. For immunization, sheep were primed with a pcDNA plasmid (pcDNA-env) encoding the Env glycoproteins of MVV and boosted with combined pcDNA-env and pCR3.1-IFN-gamma plasmid inoculations. The pcDNA plasmid used in the control group contained the lacZ coding sequences instead of the env gene. Within a month post-challenge, the viral load in the vaccinated group was lower (p < or = 0.05) and virus was only detected transiently compared with the control group. Furthermore, 2 months later, neutralizing antibodies (NtAb) were detected in all the control animals and none of the vaccinated animals (p < or = 0.01). These results demonstrated a significant early protective effect of this immunization strategy against MVV infection that restricts the virus replication following challenge in the absence of NtAb production. This vaccine protective effect against MVV infection disappeared after two years post-challenge, when active replication of MVV challenge strain was observed. Protection conferred by the vaccine could not be explained by OLA DRB1 allele or genotype differences. Most of the individuals were DRB1 heterozygous and none was totally resistant to infection.

  16. Genome-Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor

    PubMed Central

    Malicorne, Sébastien; Vernochet, Cécile; Cornelis, Guillaume; Mulot, Baptiste; Delsuc, Frédéric; Heidmann, Odile

    2016-01-01

    ABSTRACT Retroviruses enter host cells through the interaction of their envelope (Env) protein with a cell surface receptor, which triggers the fusion of viral and cellular membranes. The sodium-dependent neutral amino acid transporter ASCT2 is the common receptor of the large RD114 retrovirus interference group, whose members display frequent env recombination events. Germ line retrovirus infections have led to numerous inherited endogenous retroviruses (ERVs) in vertebrate genomes, which provide useful insights into the coevolutionary history of retroviruses and their hosts. Rare ERV-derived genes display conserved viral functions, as illustrated by the fusogenic syncytin env genes involved in placentation. Here, we searched for functional env genes in the nine-banded armadillo (Dasypus novemcinctus) genome and identified dasy-env1.1, which clusters with RD114 interference group env genes and with two syncytin genes sharing ASCT2 receptor usage. Using ex vivo pseudotyping and cell-cell fusion assays, we demonstrated that the Dasy-Env1.1 protein is fusogenic and can use both human and armadillo ASCT2s as receptors. This gammaretroviral env gene belongs to a provirus with betaretrovirus-like features, suggesting acquisition through recombination. Provirus insertion was found in several Dasypus species, where it has not reached fixation, whereas related family members integrated before diversification of the genus Dasypus >12 million years ago (Mya). This newly described ERV lineage is potentially useful as a population genetic marker. Our results extend the usage of ASCT2 as a retrovirus receptor to the mammalian clade Xenarthra and suggest that the acquisition of an ASCT2-interacting env gene is a major selective force driving the emergence of numerous chimeric viruses in vertebrates. IMPORTANCE Retroviral infection is initiated by the binding of the viral envelope glycoprotein to a host cell receptor(s), triggering membrane fusion. Ancient germ line infections

  17. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  18. Major Sperm Protein Genes from Globodera rostochiensis

    PubMed Central

    Novitski, Charles E.; Brown, Shiela; Chen, Ru; Corner, Adam S.; Atkinson, Howard J.; McPherson, Michael J.

    1993-01-01

    Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis. PMID:19279808

  19. Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

    PubMed Central

    Kesavardhana, Sannula

    2014-01-01

    ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers

  20. Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation.

    PubMed

    Ferris, Hedda U; Coles, Murray; Lupas, Andrei N; Hartmann, Marcus D

    2014-06-01

    Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.

  1. Macrophage entry mediated by HIV Envs from brain and lymphoid tissues is determined by the capacity to use low CD4 levels and overall efficiency of fusion

    SciTech Connect

    Thomas, Elaine R.; Dunfee, Rebecca L.; Stanton, Jennifer; Bogdan, Derek; Taylor, Joann; Kunstman, Kevin; Bell, Jeanne E.; Wolinsky, Steven M.; Gabuzda, Dana . E-mail: dana_gabuzda@dfci.harvard.edu

    2007-03-30

    HIV infects macrophages and microglia in the central nervous system (CNS), which express lower levels of CD4 than CD4+ T cells in peripheral blood. To investigate mechanisms of HIV neurotropism, full-length env genes were cloned from autopsy brain and lymphoid tissues from 4 AIDS patients with HIV-associated dementia (HAD). Characterization of 55 functional Env clones demonstrated that Envs with reduced dependence on CD4 for fusion and viral entry are more frequent in brain compared to lymphoid tissue. Envs that mediated efficient entry into macrophages were frequent in brain but were also present in lymphoid tissue. For most Envs, entry into macrophages correlated with overall fusion activity at all levels of CD4 and CCR5. gp160 nucleotide sequences were compartmentalized in brain versus lymphoid tissue within each patient. Proline at position 308 in the V3 loop of gp120 was associated with brain compartmentalization in 3 patients, but mutagenesis studies suggested that P308 alone does not contribute to reduced CD4 dependence or macrophage-tropism. These results suggest that HIV adaptation to replicate in the CNS selects for Envs with reduced CD4 dependence and increased fusion activity. Macrophage-tropic Envs are frequent in brain but are also present in lymphoid tissues of AIDS patients with HAD, and entry into macrophages in the CNS and other tissues is dependent on the ability to use low receptor levels and overall efficiency of fusion.

  2. Transcriptional enhancer from milk protein genes

    DOEpatents

    Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  3. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  4. RPG: the Ribosomal Protein Gene database

    PubMed Central

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes. PMID:14681386

  5. RPG: the Ribosomal Protein Gene database.

    PubMed

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes.

  6. Amelogenesis Imperfecta; Genes, Proteins, and Pathways

    PubMed Central

    Smith, Claire E. L.; Poulter, James A.; Antanaviciute, Agne; Kirkham, Jennifer; Brookes, Steven J.; Inglehearn, Chris F.; Mighell, Alan J.

    2017-01-01

    Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the

  7. Superiority in Rhesus Macaques of Targeting HIV-1 Env Gp140 to CD40 Versus LOX-1 in Combination with Replication Competent NYVAC-KC for Induction of Env-Specific Antibody and T Cell Responses.

    PubMed

    Zurawski, Gerard; Shen, Xiaoying; Zurawski, Sandra; Tomaras, Georgia D; Montefiori, David C; Roederer, Mario; Ferrari, Guido; Lacabaratz, Christine; Klucar, Peter; Wang, Zhiqing; Foulds, Kathryn E; Kao, Shing-Fen; Yu, Xuesong; Sato, Alicia; Yates, Nicole L; LaBranche, Celia; Stanfield-Oakley, Sherry; Kibler, Karen; Jacobs, Bertram; Salazar, Andres; Self, Steve; Fulp, Jimmy; Gottardo, Raphael; Galmin, Lindsey; Weiss, Deborah; Cristillo, Anthony; Pantaleo, Giuseppe; Levy, Yves

    2017-02-15

    We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in Rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly ICLC adjuvant either alone or co-administered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to P =0.01) than a group without poly ICLC. The responses were robust, cross-reactive, and contained antibodies specific to multiple epitopes within gp140 including the C1, C2, V1-3, C4, C5, and gp41 immuno-dominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to Tier 1 viruses and antibody dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4(+) and CD8(+) T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines + poly ICLC. Together, these results indicate that prime/boost immunization via NYVAC-KC and either αCD40.Env gp140/poly ICLC or αLOX-1.Env gp140/poly ICLC induced balanced antibody and T cell responses against HIV-1 Env. Co-administration of NYVAC-KC with the DC-targeting vaccines increased T cell responses, but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, compared to LOX-1, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability.IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells

  8. A safe packaging line for gene transfer: separating viral genes on two different plasmids.

    PubMed Central

    Markowitz, D; Goff, S; Bank, A

    1988-01-01

    A retrovirus packaging cell line was constructed by using portions of the Moloney murine leukemia virus in which the gag, pol, and env genes of the helper virus were separated onto two different plasmids and in which the psi packaging signal and 3' long terminal repeat were removed. The plasmid containing the gag and pol genes and the plasmid containing the env gene were cotransfected into NIH 3T3 cells. Clones that produced high levels of reverse transcriptase and env protein were tested for their ability to package the replication-defective retrovirus vectors delta neo and N2. One of the gag-pol and env clones (GP+E-86) was able to transfer G418 resistance to recipient cells at a titer of as high as 1.7 X 10(5) when it was used to package delta neo and as high as 4 X 10(6) when it was used to package N2. Supernatants of clones transfected with the intact parent gag-pol-env plasmid 3P0 had comparable titers (as high as 6.5 X 10(4) with delta neo; as high as 1.7 X 10(5) with N2). Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus. These results suggest that gag, pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer. Images PMID:2831375

  9. Repeated Vaccination of Cows with HIV Env gp140 during Subsequent Pregnancies Elicits and Sustains an Enduring Strong Env-Binding and Neutralising Antibody Response

    PubMed Central

    Center, Rob J.; Gonelli, Christopher; Muller, Brian; Mackenzie, Charlene; Khoury, Georges; Lichtfuss, Marit; Rawlin, Grant; Purcell, Damian F. J.

    2016-01-01

    An important feature of a potential vaccine against HIV is the production of broadly neutralising antibodies (BrNAbs) capable of potentially blocking infectivity of a diverse array of HIV strains. BrNAbs naturally arise in some HIV infected individuals after several years of infection and their serum IgG can neutralise various HIV strains across different subtypes. We previously showed that vaccination of cows with HIV gp140 AD8 trimers resulted in a high titre of serum IgG against HIV envelope (Env) that had strong BrNAb activity. These polyclonal BrNAbs concentrated into the colostrum during the late stage of pregnancy and can be harvested in vast quantities immediately after calving. In this study, we investigated the effect of prolonged HIV gp140 vaccination on bovine colostrum IgG HIV Env-binding and BrNAb activity over subsequent pregnancies. Repeated immunisation led to a maintained high titre of HIV Env specific IgG in the colostrum batches, but this did not increase through repeated cycles. Colostrum IgG from all batches also strongly competed with sCD4 binding to gp140 Env trimer and with human-derived monoclonal VRC01 and b12 BrNAbs that bind the CD4 binding site (CD4bs). Furthermore, competition neutralisation assays using RSC3 Env gp120 protein core and a derivative CD4bs mutant, RSC3 Δ371I/P363N, showed that CD4bs neutralising antibodies contribute to the neutralising activity of all batches of purified bovine colostrum IgG. This result indicates that the high IgG titre/avidity of anti-CD4bs antibodies with BrNAb activity was achieved during the first year of vaccination and was sustained throughout the years of repeated vaccinations in the cow tested. Although IgG of subsequent colostrum batches may have a higher avidity towards the CD4bs, the overall breadth in neutralisation was not enhanced. This implies that the boosting vaccinations over 4 years elicited a polyclonal antibody response that maintained the proportion of both neutralising and non

  10. Repeated Vaccination of Cows with HIV Env gp140 during Subsequent Pregnancies Elicits and Sustains an Enduring Strong Env-Binding and Neutralising Antibody Response.

    PubMed

    Heydarchi, Behnaz; Center, Rob J; Gonelli, Christopher; Muller, Brian; Mackenzie, Charlene; Khoury, Georges; Lichtfuss, Marit; Rawlin, Grant; Purcell, Damian F J

    2016-01-01

    An important feature of a potential vaccine against HIV is the production of broadly neutralising antibodies (BrNAbs) capable of potentially blocking infectivity of a diverse array of HIV strains. BrNAbs naturally arise in some HIV infected individuals after several years of infection and their serum IgG can neutralise various HIV strains across different subtypes. We previously showed that vaccination of cows with HIV gp140 AD8 trimers resulted in a high titre of serum IgG against HIV envelope (Env) that had strong BrNAb activity. These polyclonal BrNAbs concentrated into the colostrum during the late stage of pregnancy and can be harvested in vast quantities immediately after calving. In this study, we investigated the effect of prolonged HIV gp140 vaccination on bovine colostrum IgG HIV Env-binding and BrNAb activity over subsequent pregnancies. Repeated immunisation led to a maintained high titre of HIV Env specific IgG in the colostrum batches, but this did not increase through repeated cycles. Colostrum IgG from all batches also strongly competed with sCD4 binding to gp140 Env trimer and with human-derived monoclonal VRC01 and b12 BrNAbs that bind the CD4 binding site (CD4bs). Furthermore, competition neutralisation assays using RSC3 Env gp120 protein core and a derivative CD4bs mutant, RSC3 Δ371I/P363N, showed that CD4bs neutralising antibodies contribute to the neutralising activity of all batches of purified bovine colostrum IgG. This result indicates that the high IgG titre/avidity of anti-CD4bs antibodies with BrNAb activity was achieved during the first year of vaccination and was sustained throughout the years of repeated vaccinations in the cow tested. Although IgG of subsequent colostrum batches may have a higher avidity towards the CD4bs, the overall breadth in neutralisation was not enhanced. This implies that the boosting vaccinations over 4 years elicited a polyclonal antibody response that maintained the proportion of both neutralising and non

  11. HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV.

    PubMed

    Madhavi, Vijaya; Wines, Bruce D; Amin, Janaki; Emery, Sean; Lopez, Ester; Kelleher, Anthony; Center, Rob J; Hogarth, P Mark; Chung, Amy W; Kent, Stephen J; Stratov, Ivan

    2017-09-15

    Studying HIV-infected individuals who control HIV replication (elite controllers [ECs]) enables exploration of effective anti-HIV immunity. HIV Env-specific and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection, but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses in sera were studied using a novel enzyme-linked immunosorbent assay (ELISA)-based dimeric recombinant soluble FcγRIIIa (rsFcγRIIIa)-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays, and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcγRIIIa, activating NK cells, and mediating granzyme B activity (all P < 0.01) than viremic subjects. ECs also had higher levels of antibodies against a C-terminal 13-mer Vpu peptide capable of mediating FcγRIIIa binding and NK cell activation than viremic subjects (both P < 0.05). Our data associate Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control.IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove to be of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines. Copyright © 2017 American Society for Microbiology.

  12. Stress Genes and Proteins in the Archaea

    PubMed Central

    Macario, Alberto J. L.; Lange, Marianne; Ahring, Birgitte K.; De Macario, Everly Conway

    1999-01-01

    The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of ∼60 kDa. These

  13. Structural and functional studies of the HAMP domain of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli.

    PubMed

    Kishii, Ryuta; Falzon, Liliana; Yoshida, Takeshi; Kobayashi, Hiroshi; Inouye, Masayori

    2007-09-07

    The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg(180)-Thr(235)) with the R218K mutation (termed L(RK)) or with L(RK) connected with domain A (Arg(180)-Arg(289)) (termed LA(RK)) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L(RK) and LA(RK) proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala(193) in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA(RK)(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA(RK)(A193L) became substantially lower than that of LA(RK) or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929-940).

  14. Mining disease genes using integrated protein-protein interaction and gene-gene co-regulation information.

    PubMed

    Li, Jin; Wang, Limei; Guo, Maozu; Zhang, Ruijie; Dai, Qiguo; Liu, Xiaoyan; Wang, Chunyu; Teng, Zhixia; Xuan, Ping; Zhang, Mingming

    2015-01-01

    In humans, despite the rapid increase in disease-associated gene discovery, a large proportion of disease-associated genes are still unknown. Many network-based approaches have been used to prioritize disease genes. Many networks, such as the protein-protein interaction (PPI), KEGG, and gene co-expression networks, have been used. Expression quantitative trait loci (eQTLs) have been successfully applied for the determination of genes associated with several diseases. In this study, we constructed an eQTL-based gene-gene co-regulation network (GGCRN) and used it to mine for disease genes. We adopted the random walk with restart (RWR) algorithm to mine for genes associated with Alzheimer disease. Compared to the Human Protein Reference Database (HPRD) PPI network alone, the integrated HPRD PPI and GGCRN networks provided faster convergence and revealed new disease-related genes. Therefore, using the RWR algorithm for integrated PPI and GGCRN is an effective method for disease-associated gene mining.

  15. Susceptibility of domestic animals to a pseudotype virus bearing RD-114 virus envelope protein.

    PubMed

    Miyaho, Rie Nakaoka; Nakagawa, So; Hashimoto-Gotoh, Akira; Nakaya, Yuki; Shimode, Sayumi; Sakaguchi, Shoichi; Yoshikawa, Rokusuke; Takahashi, Mahoko Ueda; Miyazawa, Takayuki

    2015-08-10

    Retroviral vectors are used for gene transduction into cells and have been applied to gene therapy. Retroviral vectors using envelope protein (Env) of RD-114 virus, a feline endogenous retrovirus, have been used for gene transduction. In this study, we investigated the susceptibility to RD-114 Env-pseudotyped virus in twelve domestic animals including cattle, sheep, horse, pig, dog, cat, ferret, mink, rabbit, rat, mouse, and quail. Comparison of nucleotide sequences of ASCT2 (SLC1A5), a receptor of RD-114 virus, in 10 mammalian and 2 avian species revealed that insertion and deletion events at the region C of ASCT2 where RD-114 viral Env interacts occurred independently in the mouse and rat lineage and in the chicken and quail lineage. By the pseudotype virus infection assay, we found that RD-114 Env-pseudotyped virus could efficiently infect all cell lines except those from mouse and rat. Furthermore, we confirmed that bovine ASCT2 (bASCT2) functions as a receptor for RD-114 virus infection. We also investigated bASCT2 mRNA expression in cattle tissues and found that it is expressed in various tissues including lung, spleen and kidney. These results indicate that retrovirus vectors with RD-114 virus Env can be used for gene therapy in large domestic animals in addition to companion animals such as cat and dog.

  16. Persian adaptation of a questionnaire of environmental risk factors in multiple sclerosis (EnvIMS-Q).

    PubMed

    Sahraian, Mohammad Ali; Naghshineh, Hoda; Shati, Mohsen; Jahromi, Soodeh Razeghi; Rezaei, Niloofar

    2016-11-01

    It seems that gene-environment interaction play most important role in Multiple Sclerosis development. Increasing the incidence and prevalence of MS during the recent decades in the low prevalence area such as Iran is explained better by environment factors. Environmental Risk Factors in Multiple Sclerosis (the 'EnvIMS-Q') is a 6-page self-administered questionnaire for case control studies. the objectives of study are validation and adaptation of the EnvIMS-Q' then development of a Persian version for case control studies in Persian population. This questionnaire translated literally and in culturally relevant form, then content validation process was done by three groups' experts. According to giving rating to each item, each section and the whole instrument, we calculated their content validation indexes and also added some new questions and a new section to EnvIMS-Q. Finally, we analyzed repeatability of the answers within a 4 weeks interval. Relevancy and clarity indexes of all items were more than 80%. Scale relevancy index equaled 99% and scale clarity index equaled 97%. Repeatability of most items was acceptable. the use of standardized validated questionnaires will assist the researchers to perform local studies on the role of environmental factors on the basis of reliable data. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding

    PubMed Central

    Moscoso, Carlos G.; Sun, Yide; Poon, Selina; Xing, Li; Kan, Elaine; Martin, Loïc; Green, Dominik; Lin, Frank; Vahlne, Anders G.; Barnett, Susan; Srivastava, Indresh; Cheng, R. Holland

    2011-01-01

    The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120–gp41 interactions, whereas a “flat open” concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N- and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes. PMID:21444771

  18. A biosensor assay for studying ligand-membrane receptor interactions: Binding of antibodies and HIV-1 Env to chemokine receptors

    PubMed Central

    Hoffman, Trevor L.; Canziani, Gabriela; Jia, Li; Rucker, Joseph; Doms, Robert W.

    2000-01-01

    The HIV envelope (Env) protein mediates entry into cells by binding CD4 and an appropriate coreceptor, which triggers structural changes in Env that lead to fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the seven transmembrane domain chemokine receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain is an important determinant of viral tropism and pathogenesis, and virus-receptor interactions can be therapeutic targets. However, Envs from many virus strains interact with CXCR4 and CCR5 with low affinity such that direct study of this important interaction is difficult if not impossible using standard cell-surface binding techniques. We have developed an approach that makes it possible to study ligand binding to membrane proteins, including Env–coreceptor interactions, using an optical biosensor. CCR5, CXCR4, and other membrane proteins were incorporated into retrovirus particles, which were purified and attached to the biosensor surface. Binding of conformationally sensitive antibodies as well as Env to these receptors was readily detected. The equilibrium dissociation constant for the interaction between an Env derived from the prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM, explaining the difficulty in measuring this interaction using standard equilibrium binding techniques. Retroviral pseudotypes represent easily produced, stable, homogenous structures that can be used to present a wide array of single and multiple membrane-spanning proteins in a native lipid environment for biosensor studies, thus avoiding the need for detergent solubilization, purification, and reconstitution. The approach should have general applicability and can be used to correlate Env–receptor binding constants to viral tropism and pathogenesis. PMID:11005830

  19. Human endogenous retrovirus W family envelope gene activates the small conductance Ca2+-activated K+ channel in human neuroblastoma cells through CREB.

    PubMed

    Li, S; Liu, Z C; Yin, S J; Chen, Y T; Yu, H L; Zeng, J; Zhang, Q; Zhu, F

    2013-09-05

    Numerous studies have shown that human endogenous retrovirus W family (HERV-W) envelope gene (env) is related to various diseases but the underlying mechanism has remained poorly understood. Our previous study showed that there was abnormal expression of HERV-W env in sera of patients with schizophrenia. In this paper, we reported that overexpression of the HERV-W env elevated the levels of small conductance Ca(2+)-activated K(+) channel protein 3 (SK3) in human neuroblastoma cells. Using a luciferase reporter system and RNA interference method, we found that functional cAMP response element site was required for the expression of SK3 triggered by HERV-W env. In addition, it was also found that the SK3 channel was activated by HERV-W env. Further study indicated that cAMP response element-binding protein (CREB) was required for the activation of the SK3 channel. Thus, a novel signaling mechanism of how HERV-W env influences neuronal activity and contributes to mental illnesses such as schizophrenia was proposed.

  20. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  1. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  2. Increased T cell breadth and antibody response elicited in prime-boost regimen by viral vector encoded homologous SIV Gag/Env in outbred CD1 mice.

    PubMed

    Andersson, Anne-Marie Carola; Holst, Peter Johannes

    2016-12-20

    A major obstacle for the development of HIV vaccines is the virus' worldwide sequence diversity. Nevertheless, the presence of T cell epitopes within conserved regions of the virus' structural Gag protein and conserved structures in the envelope (env) sequence raises the possibility that cross-reactive responses may be induced by vaccination. In this study, the aim was to investigate the importance of antigenic match on immunodominance and breadth of obtainable T cell responses. Outbred CD1 mice were immunized with either heterologous (SIVmac239 and HIV-1 clade B consensus) or homologous (SIVmac239) gag sequences using adenovirus (Ad5) and MVA vectors. Env (SIVmac239) was co-encoded in the vectors to study the induction of antibodies, which is a primary target of current HIV vaccine designs. All three vaccines were designed as virus-encoded virus-like particle vaccines. Antibody responses were analysed by ELISA, avidity ELISA, and neutralization assay. T cell responses were determined by intracellular cytokine staining of splenocytes. The homologous Env/Gag prime-boost regimen induced higher Env binding antibodies, and induced stronger and broader Gag specific CD8+ T cell responses than the homologous Env/heterologous Gag prime-boost regimen. Homologous Env/heterologous Gag immunization resulted in selective boosting of Env specific CD8+ T cell responses and consequently a paradoxical decreased recognition of variant sequences including conserved elements of p24 Gag. These results contrast with related studies using Env or Gag as the sole antigen and suggest that prime-boost immunizations based on homologous SIVmac239 Gag inserts is an efficient component of genetic VLP vaccines-both for induction of potent antibody responses and cross-reactive CD8+ T cell responses.

  3. HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores.

    PubMed

    Monel, Blandine; Beaumont, Elodie; Vendrame, Daniela; Schwartz, Olivier; Brand, Denys; Mammano, Fabrizio

    2012-04-01

    Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.

  4. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  5. Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

    PubMed Central

    Bohl, Christopher R.; Abrahamyan, Levon G.; Wood, Charles

    2013-01-01

    The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions. PMID:23861967

  6. Recombination between feline leukemia virus subgroup B or C and endogenous env elements alters the in vitro biological activities of the viruses.

    PubMed Central

    Pandey, R; Ghosh, A K; Kumar, D V; Bachman, B A; Shibata, D; Roy-Burman, P

    1991-01-01

    An important question in feline leukemia virus (FeLV) pathogenesis is whether, as in murine leukemia virus infection, homologous recombination between the infecting FeLV and the noninfectious endogenous FeLV-like proviruses serves as a significant base for the generation of proximal pathogens. To begin an analysis of this issue, several recombinant FeLVs were produced by using two different approaches: (i) the regions of the viral envelope (env) gene of a cloned FeLV (subgroup B virus [FeLV-B], Gardner-Arnstein strain) and those of two different endogenous proviral loci were exchanged to create specific FeLV chimeras, and (ii) vectors containing endogenous env and molecularly cloned infectious FeLV-C (Sarma strain) DNA sequences were coexpressed by transfection in nonfeline cells to facilitate recombination. The results of these combined approaches showed that up to three-fourths of the envelope glycoprotein (gp70), beginning from the N-terminal end, could be replaced by endogenous FeLV sequences to produce biologically active chimeric FeLVs. The in vitro replication efficiency or cell tropism of the recombinants appeared to be influenced by the amount of gp70 sequences replaced by the endogenous partner as well as by the locus of origin of the endogenous sequences. Additionally, a characteristic biological effect, aggregation of feline T-lymphoma cells (3201B cell line), was found to be specifically induced by replicating FeLV-C or FeLV-C-based recombinants. Multiple crossover sites in the gp70 protein selected under the conditions used for coexpression were identified. The results of induced coexpression were also supported by rapid generation of FeLV recombinants when FeLV-C was used to infect the feline 3201B cell line that constitutively expresses high levels of endogenous FeLV-specific mRNAs. Furthermore, a large, highly conserved open reading frame in the pol gene of an endogenous FeLV provirus was identified. This observation, particularly in reference to

  7. Predicting gene ontology annotations of orphan GWAS genes using protein-protein interactions.

    PubMed

    Kuppuswamy, Usha; Ananthasubramanian, Seshan; Wang, Yanli; Balakrishnan, Narayanaswamy; Ganapathiraju, Madhavi K

    2014-04-03

    The number of genome-wide association studies (GWAS) has increased rapidly in the past couple of years, resulting in the identification of genes associated with different diseases. The next step in translating these findings into biomedically useful information is to find out the mechanism of the action of these genes. However, GWAS studies often implicate genes whose functions are currently unknown; for example, MYEOV, ANKLE1, TMEM45B and ORAOV1 are found to be associated with breast cancer, but their molecular function is unknown. We carried out Bayesian inference of Gene Ontology (GO) term annotations of genes by employing the directed acyclic graph structure of GO and the network of protein-protein interactions (PPIs). The approach is designed based on the fact that two proteins that interact biophysically would be in physical proximity of each other, would possess complementary molecular function, and play role in related biological processes. Predicted GO terms were ranked according to their relative association scores and the approach was evaluated quantitatively by plotting the precision versus recall values and F-scores (the harmonic mean of precision and recall) versus varying thresholds. Precisions of ~58% and ~ 40% for localization and functions respectively of proteins were determined at a threshold of ~30 (top 30 GO terms in the ranked list). Comparison with function prediction based on semantic similarity among nodes in an ontology and incorporation of those similarities in a k-nearest neighbor classifier confirmed that our results compared favorably. This approach was applied to predict the cellular component and molecular function GO terms of all human proteins that have interacting partners possessing at least one known GO annotation. The list of predictions is available at http://severus.dbmi.pitt.edu/engo/GOPRED.html. We present the algorithm, evaluations and the results of the computational predictions, especially for genes identified in

  8. Molecular mechanisms of ribosomal protein gene coregulation

    PubMed Central

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B. Franklin

    2015-01-01

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20–50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1–TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. PMID:26385964

  9. Molecular mechanisms of ribosomal protein gene coregulation.

    PubMed

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B Franklin

    2015-09-15

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. © 2015 Reja et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site

    PubMed Central

    West, Anthony P; Schamber, Michael; Gazumyan, Anna; Golijanin, Jovana; Seaman, Michael S; Fätkenheuer, Gerd; Klein, Florian; Nussenzweig, Michel C; Bjorkman, Pamela J

    2016-01-01

    HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46–derived CD4bs bNAbs, which have normal-length CDRL3s. The existence of bNAbs that combine features of VRC01-class and 8ANC131-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs. PMID:27617431

  11. An amphipathic sequence in the cytoplasmic tail of HIV-1 Env alters cell tropism and modulates viral receptor specificity.

    PubMed

    Vzorov, A N; Yang, C; Compans, R W

    2015-09-01

    The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.

  12. Protein-Protein Interaction Network and Gene Ontology

    NASA Astrophysics Data System (ADS)

    Choi, Yunkyu; Kim, Seok; Yi, Gwan-Su; Park, Jinah

    Evolution of computer technologies makes it possible to access a large amount and various kinds of biological data via internet such as DNA sequences, proteomics data and information discovered about them. It is expected that the combination of various data could help researchers find further knowledge about them. Roles of a visualization system are to invoke human abilities to integrate information and to recognize certain patterns in the data. Thus, when the various kinds of data are examined and analyzed manually, an effective visualization system is an essential part. One instance of these integrated visualizations can be combination of protein-protein interaction (PPI) data and Gene Ontology (GO) which could help enhance the analysis of PPI network. We introduce a simple but comprehensive visualization system that integrates GO and PPI data where GO and PPI graphs are visualized side-by-side and supports quick reference functions between them. Furthermore, the proposed system provides several interactive visualization methods for efficiently analyzing the PPI network and GO directedacyclic- graph such as context-based browsing and common ancestors finding.

  13. What's that gene (or protein)? Online resources for exploring functions of genes, transcripts, and proteins

    PubMed Central

    Hutchins, James R. A.

    2014-01-01

    The genomic era has enabled research projects that use approaches including genome-scale screens, microarray analysis, next-generation sequencing, and mass spectrometry–based proteomics to discover genes and proteins involved in biological processes. Such methods generate data sets of gene, transcript, or protein hits that researchers wish to explore to understand their properties and functions and thus their possible roles in biological systems of interest. Recent years have seen a profusion of Internet-based resources to aid this process. This review takes the viewpoint of the curious biologist wishing to explore the properties of protein-coding genes and their products, identified using genome-based technologies. Ten key questions are asked about each hit, addressing functions, phenotypes, expression, evolutionary conservation, disease association, protein structure, interactors, posttranslational modifications, and inhibitors. Answers are provided by presenting the latest publicly available resources, together with methods for hit-specific and data set–wide information retrieval, suited to any genome-based analytical technique and experimental species. The utility of these resources is demonstrated for 20 factors regulating cell proliferation. Results obtained using some of these are discussed in more depth using the p53 tumor suppressor as an example. This flexible and universally applicable approach for characterizing experimental hits helps researchers to maximize the potential of their projects for biological discovery. PMID:24723265

  14. New insights into the mechanism of light modulated signaling by heterotrimeric G-proteins: ENVOY acts on gna1 and gna3 and adjusts cAMP levels in Trichoderma reesei (Hypocrea jecorina)

    PubMed Central

    Tisch, Doris; Kubicek, Christian P.; Schmoll, Monika

    2011-01-01

    Sensing of environmental signals is often mediated by G-protein coupled receptors and their cognate heterotrimeric G-proteins. In Trichoderma reesei (Hypocrea jecorina) the signals transmitted via the G-protein alpha subunits GNA1 and GNA3 cause considerable modulation of cellulase transcript levels and the extent of this adjustment is dependent on the light status. We therefore intended to elucidate the underlying mechanism connecting light response and heterotrimeric G-protein signaling. Analysis of double mutant strains showed that constitutive activation of GNA1 or GNA3 in the absence of the PAS/LOV domain protein ENVOY (ENV1) leads to the phenotype of constitutive G-alpha activation in darkness. In light, however the deletion-phenotype of Δenv1 was observed with respect to growth, conidiation and cellulase gene transcription. Additionally deletion of env1 causes decreased intracellular cAMP accumulation, even upon constitutive activation of GNA1 or GNA3. While supplementation of cAMP caused an even more severe growth phenotype of all strains lacking env1 in light, addition of the phosphodiesterase inhibitor caffeine rescued the growth phenotype of these strains. ENV1 is consequently suggested to connect the light response pathway with nutrient signaling by the heterotrimeric G-protein cascade by adjusting transcript levels of gna1 and gna3 and action on cAMP levels – presumably through inhibition of a phosphodiesterase. PMID:21220037

  15. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    PubMed

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  16. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  17. Evolution of B cell analysis and Env trimer redesign.

    PubMed

    Karlsson Hedestam, Gunilla B; Guenaga, Javier; Corcoran, Martin; Wyatt, Richard T

    2017-01-01

    HIV-1 and its surface envelope glycoproteins (Env), gp120 and gp41, have evolved immune evasion strategies that render the elicitation of effective antibody responses to the functional Env entry unit extremely difficult. HIV-1 establishes chronic infection and stimulates vigorous immune responses in the human host; forcing selection of viral variants that escape cellular and antibody (Ab)-mediated immune pressure, yet possess contemporary fitness. Successful survival of fit variants through the gauntlet of the human immune system make this virus and these glycoproteins a formidable challenge to target by vaccination, requiring a systematic approach to Env mimetic immunogen design and evaluation of elicited responses. Here, we review key aspects of HIV-1 Env immunogenicity and immunogen re-design, based on experimental data generated by us and others over the past decade or more. We further provide rationale and details regarding the use of newly evolving tools to analyze B cell responses, including approaches to use next generation sequencing for antibody lineage tracing and B cell fate mapping. Together, these developments offer opportunities to address long-standing questions about the establishment of effective B cell immunity elicited by vaccination, not just against HIV-1. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Appreciating HIV-1 diversity: subtypic differences in ENV

    SciTech Connect

    Gnanakaran, S; Shen, Tongye; Lynch, Rebecca M; Derdeyn, Cynthia A

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1) group M is responsible for the current AIDS pandemic and exhibits exceedingly high levels of viral genetic diversity around the world, necessitating categorization of viruses into distinct lineages, or subtypes. These subtypes can differ by around 35% in the envelope (Env) glycoproteins of the virus, which are displayed on the surface of the virion and are targets for both neutralizing antibody and cell-mediated immune responses. This diversity reflects the remarkable ability of the virus to adapt to selective pressures, the bulk of which is applied by the host immune response, and represents a serious obstacle for developing an effective vaccine with broad coverage. Thus, it is important to understand the underlying biological consequences of inter-subtype diversity. Recent studies have revealed that the HIV-1 subtypes exhibit phenotypic differences that result from subtle differences in Env structure, particularly within the highly immunogenic V3 domain, which participates directly in viral entry. This review will therefore explore current research that describes subtypic differences in Env at the genetic and phenotypic level, focusing in particular on V3, and highlighting recent discoveries about the unique features of subtype C Env, which is the most prevalent subtype globally.

  19. A pilot study comparing the development of EIAV Env-specific antibodies induced by DNA/recombinant vaccinia-vectored vaccines and an attenuated Chinese EIAV vaccine.

    PubMed

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2012-12-01

    Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAV(FDDV). Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure.

  20. A Pilot Study Comparing the Development of EIAV Env-Specific Antibodies Induced by DNA/Recombinant Vaccinia-Vectored Vaccines and an Attenuated Chinese EIAV Vaccine

    PubMed Central

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian

    2012-01-01

    Abstract Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAVFDDV. Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure. PMID:23171359

  1. Enhanced cell surface expression, immunogenicity and genetic stability resulting from a spontaneous truncation of HIV Env expressed by a recombinant MVA

    SciTech Connect

    Wyatt, Linda S. Belyakov, Igor M.; Earl, Patricia L.; Berzofsky, Jay A.; Moss, Bernard

    2008-03-15

    During propagation of modified vaccinia virus Ankara (MVA) encoding HIV 89.6 Env, a few viral foci stained very prominently. Virus cloned from such foci replicated to higher titers than the parent and displayed enhanced genetic stability on passage. Sequence analysis showed a single nucleotide deletion in the 89.6 env gene of the mutant that caused a frame shift and truncation of 115 amino acids from the cytoplasmic domain. The truncated Env was more highly expressed on the cell surface, induced higher antibody responses than the full-length Env, reacted with HIV neutralizing monoclonal antibodies and mediated CD4/co-receptor-dependent fusion. Intramuscular (IM), intradermal (ID) needleless, and intrarectal (IR) catheter inoculations gave comparable serum IgG responses. However, intraoral (IO) needleless injector route gave the highest IgA in lung washings and IR gave the highest IgA and IgG responses in fecal extracts. Induction of CTL responses in the spleens of individual mice as assayed by intracellular cytokine staining was similar with both the full-length and truncated Env constructs. Induction of acute and memory CTL in the spleens of mice immunized with the truncated Env construct by ID, IO, and IR routes was comparable and higher than by the IM route, but only the IR route induced CTL in the gut-associated lymphoid tissue. Thus, truncation of Env enhanced genetic stability as well as serum and mucosal antibody responses, suggesting the desirability of a similar modification in MVA-based candidate HIV vaccines.

  2. Identification of a HERV-K env surface peptide highly recognized in Rheumatoid Arthritis (RA) patients: a cross-sectional case-control study.

    PubMed

    Mameli, G; Erre, G L; Caggiu, E; Mura, S; Cossu, D; Bo, M; Cadoni, M L; Piras, A; Mundula, N; Colombo, E; Buscetta, G; Passiu, G; Sechi, L A

    2017-07-01

    Endogenous retroviruses (HERV) are believed to be pathogenic in several autoimmune diseases. Among them, HERV-K viruses have been reported recently to be involved in the pathogenesis of rheumatoid arthritis (RA). In this study we have explored the role of humoral immune response against HERV-K as a potential pathogenetic mechanism in RA. Four different peptides from the extracellular portion of the env protein of HERV-K (env-su19-37 , env-su109-126 , env-su164-186 , env-su209-226 ) were selected by bioinformatic analysis on the basis of their putative immunogenicity. Indirect enzyme-linked immunosorbent assay (ELISA) was then carried out to quantify antibodies against those peptides on blood samples of 70 consecutive RA patients and 71 healthy controls (HC). Differences between the two groups were analysed using the Mann-Whitney test. Potential correlations between RA laboratory, clinical descriptors and immunoglobulin (Ig)G levels were explored by bivariate regression analysis. Serum autoantibodies against one of four tested peptides of HERV-K (env-su19-37 ) were significantly higher in RA than in HC (19 versus 3%, P = 0·0025). Subgroup analysis showed no association between anti-HERV-K peptide humoral response and clinical, serological and clinimetric RA disease descriptors. Serum from RA patients in our series reacted significantly against HERV-K env-su19-37 peptide in comparison to the general population suggesting a role for the HERV-K- related, secondary antigenic-driven immune response in the pathogenesis of RA. Further studies are needed to confirm these results and to explore the role of this HERV-K surface peptide as a potential therapeutic target. © 2017 British Society for Immunology.

  3. Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein

    PubMed Central

    Cheynet, Valérie; Oriol, Guy; Mallet, François

    2006-01-01

    The cellular HERV-W envelope/syncytin-1 protein, encoded by the envelope gene of the ERVWE1 proviral locus is a fusogenic glycoprotein probably involved in the formation of the placental syncytiotrophoblast layer. Syncytin-1-induced in vitro cell-cell fusion is dependent on the interaction with hASCT2. As no receptor binding domain has been clearly defined in the SU of neither the HERV-W Env nor the retroviruses of the same interference group, we designed an in vitro binding assay to evaluate the interaction of the HERV-W envelope with the hASCT2 receptor. Using truncated HERV-W SU subunits, a region consisting of the N-terminal 124 amino acids of the mature SU glycoprotein was determined as the minimal receptor-binding domain. This domain contains several sub-domains which are poorly conserved among retroviruses of this interference group but a region of 18 residus containing the SDGGGX2DX2R conserved motif was proved to be essential for syncytin-1-hASCT2 interaction. PMID:16820059

  4. Nucleotide sequence of the coat protein gene of canine parvovirus.

    PubMed Central

    Rhode, S L

    1985-01-01

    The nucleotide sequence of the canine parvovirus (CPV2) from map units 33 to 95 has been determined. This includes the entire coat protein gene and noncoding sequences at the 3' end of the gene, exclusive of the terminal inverted repeat. The predicted capsid protein structures are discussed and compared with those of the rodent parvoviruses H-1 and MVM. PMID:3989914

  5. Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity

    PubMed Central

    Ochsenbauer-Jambor, Christina; Miller, David C.; Roberts, Charles R.; Rhee, Sung S.; Hunter, Eric

    2001-01-01

    The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to env gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed. PMID:11689636

  6. Extracting synonymous gene and protein terms from biological literature.

    PubMed

    Yu, Hong; Agichtein, Eugene

    2003-01-01

    Genes and proteins are often associated with multiple names. More names are added as new functional or structural information is discovered. Because authors can use any one of the known names for a gene or protein, information retrieval and extraction would benefit from identifying the gene and protein terms that are synonyms of the same substance. We have explored four complementary approaches for extracting gene and protein synonyms from text, namely the unsupervised, partially supervised, and supervised machine-learning techniques, as well as the manual knowledge-based approach. We report results of a large scale evaluation of these alternatives over an archive of biological journal articles. Our evaluation shows that our extraction techniques could be a valuable supplement to resources such as SWISSPROT, as our systems were able to capture gene and protein synonyms not listed in the SWISSPROT database.

  7. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  8. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  9. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2016-02-02

    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  10. Automatically identifying gene/protein terms in MEDLINE abstracts.

    PubMed

    Yu, Hong; Hatzivassiloglou, Vasileios; Rzhetsky, Andrey; Wilbur, W John

    2002-01-01

    Natural language processing (NLP) techniques are used to extract information automatically from computer-readable literature. In biology, the identification of terms corresponding to biological substances (e.g., genes and proteins) is a necessary step that precedes the application of other NLP systems that extract biological information (e.g., protein-protein interactions, gene regulation events, and biochemical pathways). We have developed GPmarkup (for "gene/protein-full name mark up"), a software system that automatically identifies gene/protein terms (i.e., symbols or full names) in MEDLINE abstracts. As a part of marking up process, we also generated automatically a knowledge source of paired gene/protein symbols and full names (e.g., LARD for lymphocyte associated receptor of death) from MEDLINE. We found that many of the pairs in our knowledge source do not appear in the current GenBank database. Therefore our methods may also be used for automatic lexicon generation. GPmarkup has 73% recall and 93% precision in identifying and marking up gene/protein terms in MEDLINE abstracts. A random sample of gene/protein symbols and full names and a sample set of marked up abstracts can be viewed at http://www.cpmc.columbia.edu/homepages/yuh9001/GPmarkup/. Contact. hy52@columbia.edu. Voice: 212-939-7028; fax: 212-666-0140.

  11. Gene composer: database software for protein construct design, codon engineering, and gene synthesis.

    PubMed

    Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

    2009-04-21

    To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease

  12. Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

    PubMed Central

    Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

    2009-01-01

    Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis

  13. Variation in simian immunodeficiency virus env is confined to V1 and V4 during progression to simian AIDS.

    PubMed Central

    Overbaugh, J; Rudensey, L M; Papenhausen, M D; Benveniste, R E; Morton, W R

    1991-01-01

    We have monitored changes in the simian immunodeficiency virus (SIV) envelope (env) gene in two macaques which developed AIDS after inoculation with a molecular clone of SIV. As the animals progressed to AIDS, selection occurred for viruses with variation in two discrete regions (V1 and V4) but not for viruses with changes in the region of SIV env that corresponds to the immunodominant, V3 loop of human immunodeficiency virus. Within the highly variable domains, the vast majority of nucleotide changes encoded an amino acid change (98%), suggesting that these envelope variants had evolved as a result of phenotypic selection. Analysis of the biological properties of these variants, which have been selected for in the host, may be useful in defining the mechanisms underlying viral persistence and progression to simian AIDS. PMID:1942255

  14. Measles virus P gene codes for two proteins.

    PubMed Central

    Bellini, W J; Englund, G; Rozenblatt, S; Arnheiter, H; Richardson, C D

    1985-01-01

    The entirety of the phosphoprotein gene of measles virus has been sequenced. The gene is composed of 1,657 nucleotides and specifies a 507-amino-acid protein (P). A second overlapping reading frame was predicted from the sequence and specifies a 186-amino-acid protein (C). Through the use of antisynthetic peptide antibodies, we show that both proteins are expressed in virally infected cells. Both proteins are expressed from a functionally bicistronic mRNA through independent initiation of ribosomes at the respective AUG codons. Using immunofluorescent microscopy, we localized the C protein in the nucleus and in cytoplasmic inclusions within the infected cells. Images PMID:3882996

  15. Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

    PubMed

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M; Fenouillet, Emmanuel

    2010-04-30

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

  16. Structure and immune recognition of trimeric prefusion HIV-1 Env

    PubMed Central

    Pancera, Marie; Zhou, Tongqing; Druz, Aliaksandr; Georgiev, Ivelin S.; Soto, Cinque; Gorman, Jason; Huang, Jinghe; Acharya, Priyamvada; Chuang, Gwo-Yu; Ofek, Gilad; Stewart-Jones, Guillaume B. E.; Stuckey, Jonathan; Bailer, Robert T.; Joyce, M. Gordon; Louder, Mark K.; Tumba, Nancy; Yang, Yongping; Zhang, Baoshan; Cohen, Myron S.; Haynes, Barton F.; Mascola, John R.; Morris, Lynn; Munro, James B.; Blanchard, Scott C.; Mothes, Walther; Connors, Mark; Kwong, Peter D.

    2015-01-01

    The HIV-1-envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a postfusion state. As the sole viral antigen on the HIV-1-virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5-Å resolution for an HIV-1-Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the prefusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Prefusion gp41 encircles N- and C-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry likely involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the prefusion closed spike: we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation. PMID:25296255

  17. T-Cell Immune Responses Against Env from CRF12_BF and Subtype B HIV-1 Show High Clade-Specificity that Can Be Overridden by Multiclade Immunizations

    PubMed Central

    Mónaco, Daniela C.; Rodríguez, Ana M.; Pascutti, María F.; Carobene, Mauricio; Falivene, Juliana; Gómez, Alejandro; Maeto, Cynthia; Turk, Gabriela; Nájera, José L.; Esteban, Mariano; Gherardi, M. Magdalena

    2011-01-01

    Background The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. Methodology/Principal Findings As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. Conclusions/Significance This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate. PMID:21364754

  18. The Classical Arabinogalactan Protein Gene Family of Arabidopsis

    PubMed Central

    Schultz, Carolyn J.; Johnson, Kim L.; Currie, Graeme; Bacic, Antony

    2000-01-01

    Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides—AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach. PMID:11006345

  19. Global Panel of HIV-1 Env Reference Strains for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

    PubMed Central

    deCamp, Allan; Hraber, Peter; Bailer, Robert T.; Seaman, Michael S.; Ochsenbauer, Christina; Kappes, John; Gottardo, Raphael; Edlefsen, Paul; Self, Steve; Tang, Haili; Greene, Kelli; Gao, Hongmei; Daniell, Xiaoju; Sarzotti-Kelsoe, Marcella; Gorny, Miroslaw K.; Zolla-Pazner, Susan; LaBranche, Celia C.; Mascola, John R.; Korber, Bette T.

    2014-01-01

    ABSTRACT Standardized assessments of HIV-1 vaccine-elicited neutralizing antibody responses are complicated by the genetic and antigenic variability of the viral envelope glycoproteins (Envs). To address these issues, suitable reference strains are needed that are representative of the global epidemic. Several panels have been recommended previously, but no clear answers have been available on how many and which strains are best suited for this purpose. We used a statistical model selection method to identify a global panel of reference Env clones from among 219 Env-pseudotyped viruses assayed in TZM-bl cells with sera from 205 HIV-1-infected individuals. The Envs and sera were sampled globally from diverse geographic locations and represented all major genetic subtypes and circulating recombinant forms of the virus. Assays with a panel size of only nine viruses adequately represented the spectrum of HIV-1 serum neutralizing activity seen with the larger panel of 219 viruses. An optimal panel of nine viruses was selected and augmented with three additional viruses for greater genetic and antigenic coverage. The spectrum of HIV-1 serum neutralizing activity seen with the final 12-virus panel closely approximated the activity seen with subtype-matched viruses. Moreover, the final panel was highly sensitive for detection of many of the known broadly neutralizing antibodies. For broader assay applications, all 12 Env clones were converted to infectious molecular clones using a proviral backbone carrying a Renilla luciferase reporter gene (Env.IMC.LucR viruses). This global panel should facilitate highly standardized assessments of vaccine-elicited neutralizing antibodies across multiple HIV-1 vaccine platforms in different parts of the world. IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary genetic variability of the virus, where most variation occurs in the viral envelope glycoproteins that are the sole targets for neutralizing antibodies

  20. Evolutionary conservation and diversification of Rh family genes and proteins

    PubMed Central

    Huang, Cheng-Han; Peng, Jianbin

    2005-01-01

    Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{3}/{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document}, whereas others think that Rh proteins transport CO2 and Amt proteins NH3. In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution. PMID:16227429

  1. Evolutionary conservation and diversification of Rh family genes and proteins.

    PubMed

    Huang, Cheng-Han; Peng, Jianbin

    2005-10-25

    Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, NH(3)/NH(4)(+), whereas others think that Rh proteins transport CO(2) and Amt proteins NH(3). In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution.

  2. Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA.

    PubMed

    Julien, Jean-Philippe; Lee, Peter S; Wilson, Ian A

    2012-11-01

    Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.

  3. Matrix Gla protein and osteocalcin: from gene duplication to neofunctionalization.

    PubMed

    Cancela, M Leonor; Laizé, Vincent; Conceição, Natércia

    2014-11-01

    Osteocalcin (OC or bone Gla protein, BGP) and matrix Gla protein (MGP) are two members of the growing family of vitamin K-dependent (VKD) proteins. They were the first VKD proteins found not to be involved in coagulation and synthesized outside the liver. Both proteins were isolated from bone although it is now known that only OC is synthesized by bone cells under normal physiological conditions, but since both proteins can bind calcium and hydroxyapatite, they can also accumulate in bone. Both OC and MGP share similar structural features, both in terms of protein domains and gene organization. OC gene is likely to have appeared from MGP through a tandem gene duplication that occurred concomitantly with the appearance of the bony vertebrates. Despite their relatively close relationship and the fact that both can bind calcium and affect mineralization, their functions are not redundant and they also have other unrelated functions. Interestingly, these two proteins appear to have followed quite different evolutionary strategies in order to acquire novel functionalities, with OC following a gene duplication strategy while MGP variability was obtained mostly by the use of multiple promoters and alternative splicing, leading to proteins with additional functional characteristics and alternative gene regulatory pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. De Novo Origin of Human Protein-Coding Genes

    PubMed Central

    Wu, Dong-Dong; Irwin, David M.; Zhang, Ya-Ping

    2011-01-01

    The de novo origin of a new protein-coding gene from non-coding DNA is considered to be a very rare occurrence in genomes. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee. The functionality of these genes is supported by both transcriptional and proteomic evidence. RNA–seq data indicate that these genes have their highest expression levels in the cerebral cortex and testes, which might suggest that these genes contribute to phenotypic traits that are unique to humans, such as improved cognitive ability. Our results are inconsistent with the traditional view that the de novo origin of new genes is very rare, thus there should be greater appreciation of the importance of the de novo origination of genes. PMID:22102831

  5. Precursor Polypeptides to Structural Proteins of Visna Virus

    PubMed Central

    Vigne, Robert; Filippi, Pierre; Quérat, Gilles; Sauze, Nicole; Vitu, Christian; Russo, Pierre; Delori, Pierre

    1982-01-01

    Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55gag). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55gag and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55gag, is a likely precursor to the viral reverse transcriptase (Pr150gag-pol). Pr55gag and Pr150gag-pol are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150env). Pulse-chase experiments indicated that gPr150env matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150env, which accumulated in the presence of 2-deoxy

  6. Identification of oral cancer related candidate genes by integrating protein-protein interactions, gene ontology, pathway analysis and immunohistochemistry.

    PubMed

    Kumar, Ravindra; Samal, Sabindra K; Routray, Samapika; Dash, Rupesh; Dixit, Anshuman

    2017-05-30

    In the recent years, bioinformatics methods have been reported with a high degree of success for candidate gene identification. In this milieu, we have used an integrated bioinformatics approach assimilating information from gene ontologies (GO), protein-protein interaction (PPI) and network analysis to predict candidate genes related to oral squamous cell carcinoma (OSCC). A total of 40973 PPIs were considered for 4704 cancer-related genes to construct human cancer gene network (HCGN). The importance of each node was measured in HCGN by ten different centrality measures. We have shown that the top ranking genes are related to a significantly higher number of diseases as compared to other genes in HCGN. A total of 39 candidate oral cancer target genes were predicted by combining top ranked genes and the genes corresponding to significantly enriched oral cancer related GO terms. Initial verification using literature and available experimental data indicated that 29 genes were related with OSCC. A detailed pathway analysis led us to propose a role for the selected candidate genes in the invasion and metastasis in OSCC. We further validated our predictions using immunohistochemistry (IHC) and found that the gene FLNA was upregulated while the genes ARRB1 and HTT were downregulated in the OSCC tissue samples.

  7. The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity.

    PubMed

    Wang, Yimeng; Pan, Qinghua; Ding, Shilei; Wang, Zhen; Yu, Jingyou; Finzi, Andrés; Liu, Shan-Lu; Liang, Chen

    2017-04-01

    Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition.IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3

  8. GeneSense: a new approach for human gene annotation integrated with protein-protein interaction networks.

    PubMed

    Chen, Zhongzhong; Zhang, Tianhong; Lin, Jun; Yan, Zidan; Wang, Yongren; Zheng, Weiqiang; Weng, Kevin C

    2014-03-26

    Virtually all cellular functions involve protein-protein interactions (PPIs). As an increasing number of PPIs are identified and vast amount of information accumulated, researchers are finding different ways to interrogate the data and understand the interactions in context. However, it is widely recognized that a significant portion of the data is scattered, redundant, not considered high quality, and not readily accessible to researchers in a systematic fashion. In addition, it is challenging to identify the optimal protein targets in the current PPI networks. The GeneSense server was developed to integrate gene annotation and PPI networks in an expandable architecture that incorporates selected databases with the aim to assemble, analyze, evaluate and disseminate protein-protein association information in a comprehensive and user-friendly manner. Three network models including nodenet, leafnet and loopnet are used to identify the optimal protein targets in the complex networks. GeneSense is freely available at www.biomedsense.org/genesense.php.

  9. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  10. The KP4 killer protein gene family

    USDA-ARS?s Scientific Manuscript database

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  11. Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

    PubMed Central

    Fouda, Genevieve G. A.; Amos, Joshua D.; Wilks, Andrew B.; Pollara, Justin; Ray, Caroline A.; Chand, Anjali; Kunz, Erika L.; Liebl, Brooke E.; Whitaker, Kaylan; Carville, Angela; Smith, Shannon; Colvin, Lisa; Pickup, David J.; Staats, Herman F.; Overman, Glenn; Eutsey-Lloyd, Krissey; Parks, Robert; Chen, Haiyan; LaBranche, Celia; Barnett, Susan; Tomaras, Georgia D.; Ferrari, Guido; Montefiori, David C.; Liao, Hua-Xin; Letvin, Norman L.; Haynes, Barton F.

    2013-01-01

    We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission. PMID:23596289

  12. Identification of breast cancer candidate genes using gene co-expression and protein-protein interaction information.

    PubMed

    Yue, Zhenyu; Li, Hai-Tao; Yang, Yabing; Hussain, Sajid; Zheng, Chun-Hou; Xia, Junfeng; Chen, Yan

    2016-06-14

    Breast cancer (BC) is one of the most common malignancies that could threaten female health. As the molecular mechanism of BC has not yet been completely discovered, identification of related genes of this disease is an important area of research that could provide new insights into gene function as well as potential treatment targets. Here we used subnetwork extraction algorithms to identify novel BC related genes based on the known BC genes (seed genes), gene co-expression profiles and protein-protein interaction network. We computationally predicted seven key genes (EPHX2, GHRH, PPYR1, ALPP, KNG1, GSK3A and TRIT1) as putative genes of BC. Further analysis shows that six of these have been reported as breast cancer associated genes, and one (PPYR1) as cancer associated gene. Lastly, we developed an expression signature using these seven key genes which significantly stratified 1660 BC patients according to relapse free survival (hazard ratio [HR], 0.55; 95% confidence interval [CI], 0.46-0.65; Logrank p = 5.5e-13). The 7-genes signature could be established as a useful predictor of disease prognosis in BC patients. Overall, the identified seven genes might be useful prognostic and predictive molecular markers to predict the clinical outcome of BC patients.

  13. Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2014-01-01

    The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

  14. Effects of different promoters on the virulence and immunogenicity of a HIV-1 Env-expressing recombinant vaccinia vaccine.

    PubMed

    Isshiki, Mao; Zhang, Xianfeng; Sato, Hirotaka; Ohashi, Takashi; Inoue, Makoto; Shida, Hisatoshi

    2014-02-07

    Previously, we developed a vaccination regimen that involves priming with recombinant vaccinia virus LC16m8Δ (rm8Δ) strain followed by boosting with a Sendai virus-containing vector. This protocol induced both humoral and cellular immune responses against the HIV-1 envelope protein. The current study aims to optimize this regimen by comparing the immunogenicity and safety of two rm8Δ strains that express HIV-1 Env under the control of a moderate promoter, p7.5, or a strong promoter, pSFJ1-10. m8Δ-p7.5-JRCSFenv synthesized less gp160 but showed significantly higher growth potential than m8Δ-pSFJ-JRCSFenv. The two different rm8Δ strains induced antigen-specific immunity; however, m8Δ-pSFJ-JRCSFenv elicited a stronger anti-Env antibody response whereas m8Δ-p7.5-JRCSFenv induced a stronger Env-specific cytotoxic T lymphocyte response. Both strains were less virulent than the parental m8Δ strain, suggesting that they would be safe for use in humans. These findings indicate the vaccine can be optimized to induce favorable immune responses (either cellular or humoral), and forms the basis for the rational design of an AIDS vaccine using recombinant vaccinia as the delivery vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Tagging gene and protein names in biomedical text.

    PubMed

    Tanabe, Lorraine; Wilbur, W John

    2002-08-01

    The MEDLINE database of biomedical abstracts contains scientific knowledge about thousands of interacting genes and proteins. Automated text processing can aid in the comprehension and synthesis of this valuable information. The fundamental task of identifying gene and protein names is a necessary first step towards making full use of the information encoded in biomedical text. This remains a challenging task due to the irregularities and ambiguities in gene and protein nomenclature. We propose to approach the detection of gene and protein names in scientific abstracts as part-of-speech tagging, the most basic form of linguistic corpus annotation. We present a method for tagging gene and protein names in biomedical text using a combination of statistical and knowledge-based strategies. This method incorporates automatically generated rules from a transformation-based part-of-speech tagger, and manually generated rules from morphological clues, low frequency trigrams, indicator terms, suffixes and part-of-speech information. Results of an experiment on a test corpus of 56K MEDLINE documents demonstrate that our method to extract gene and protein names can be applied to large sets of MEDLINE abstracts, without the need for special conditions or human experts to predetermine relevant subsets. The programs are available on request from the authors.

  16. Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

    PubMed

    Green, P L; Ross, T M; Chen, I S; Pettiford, S

    1995-01-01

    Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

  17. Genes and proteins of Escherichia coli K-12.

    PubMed

    Riley, M

    1998-01-01

    GenProtEC is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins, representing groups of paralogous genes, with PAM values, percent identity of amino acids, length of alignment and percent aligned. GenProtEC can be accessed at the URL http://www.mbl.edu/html/ecoli.html

  18. Characteristics and clustering of human ribosomal protein genes

    PubMed Central

    Ishii, Kyota; Washio, Takanori; Uechi, Tamayo; Yoshihama, Maki; Kenmochi, Naoya; Tomita, Masaru

    2006-01-01

    Background The ribosome is a central player in the translation system, which in mammals consists of four RNA species and 79 ribosomal proteins (RPs). The control mechanisms of gene expression and the functions of RPs are believed to be identical. Most RP genes have common promoters and were therefore assumed to have a unified gene expression control mechanism. Results We systematically analyzed the homogeneity and heterogeneity of RP genes on the basis of their expression profiles, promoter structures, encoded amino acid compositions, and codon compositions. The results revealed that (1) most RP genes are coordinately expressed at the mRNA level, with higher signals in the spleen, lymph node dissection (LND), and fetal brain. However, 17 genes, including the P protein genes (RPLP0, RPLP1, RPLP2), are expressed in a tissue-specific manner. (2) Most promoters have GC boxes and possible binding sites for nuclear respiratory factor 2, Yin and Yang 1, and/or activator protein 1. However, they do not have canonical TATA boxes. (3) Analysis of the amino acid composition of the encoded proteins indicated a high lysine and arginine content. (4) The major RP genes exhibit a characteristic synonymous codon composition with high rates of G or C in the third-codon position and a high content of AAG, CAG, ATC, GAG, CAC, and CTG. Conclusion Eleven of the RP genes are still identified as being unique and did not exhibit at least some of the above characteristics, indicating that they may have unknown functions not present in other RP genes. Furthermore, we found sequences conserved between human and mouse genes around the transcription start sites and in the intronic regions. This study suggests certain overall trends and characteristic features of human RP genes. PMID:16504170

  19. Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins

    PubMed Central

    Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph

    2015-01-01

    pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery. PMID:26719270

  20. Calreticulin: one protein, one gene, many functions.

    PubMed Central

    Michalak, M; Corbett, E F; Mesaeli, N; Nakamura, K; Opas, M

    1999-01-01

    The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology. PMID:10567207

  1. Ranking Candidate Disease Genes from Gene Expression and Protein Interaction: A Katz-Centrality Based Approach

    PubMed Central

    Zhao, Jing; Yang, Ting-Hong; Huang, Yongxu; Holme, Petter

    2011-01-01

    Many diseases have complex genetic causes, where a set of alleles can affect the propensity of getting the disease. The identification of such disease genes is important to understand the mechanistic and evolutionary aspects of pathogenesis, improve diagnosis and treatment of the disease, and aid in drug discovery. Current genetic studies typically identify chromosomal regions associated specific diseases. But picking out an unknown disease gene from hundreds of candidates located on the same genomic interval is still challenging. In this study, we propose an approach to prioritize candidate genes by integrating data of gene expression level, protein-protein interaction strength and known disease genes. Our method is based only on two, simple, biologically motivated assumptions—that a gene is a good disease-gene candidate if it is differentially expressed in cases and controls, or that it is close to other disease-gene candidates in its protein interaction network. We tested our method on 40 diseases in 58 gene expression datasets of the NCBI Gene Expression Omnibus database. On these datasets our method is able to predict unknown disease genes as well as identifying pleiotropic genes involved in the physiological cellular processes of many diseases. Our study not only provides an effective algorithm for prioritizing candidate disease genes but is also a way to discover phenotypic interdependency, cooccurrence and shared pathophysiology between different disorders. PMID:21912686

  2. Ranking candidate disease genes from gene expression and protein interaction: a Katz-centrality based approach.

    PubMed

    Zhao, Jing; Yang, Ting-Hong; Huang, Yongxu; Holme, Petter

    2011-01-01

    Many diseases have complex genetic causes, where a set of alleles can affect the propensity of getting the disease. The identification of such disease genes is important to understand the mechanistic and evolutionary aspects of pathogenesis, improve diagnosis and treatment of the disease, and aid in drug discovery. Current genetic studies typically identify chromosomal regions associated specific diseases. But picking out an unknown disease gene from hundreds of candidates located on the same genomic interval is still challenging. In this study, we propose an approach to prioritize candidate genes by integrating data of gene expression level, protein-protein interaction strength and known disease genes. Our method is based only on two, simple, biologically motivated assumptions--that a gene is a good disease-gene candidate if it is differentially expressed in cases and controls, or that it is close to other disease-gene candidates in its protein interaction network. We tested our method on 40 diseases in 58 gene expression datasets of the NCBI Gene Expression Omnibus database. On these datasets our method is able to predict unknown disease genes as well as identifying pleiotropic genes involved in the physiological cellular processes of many diseases. Our study not only provides an effective algorithm for prioritizing candidate disease genes but is also a way to discover phenotypic interdependency, cooccurrence and shared pathophysiology between different disorders.

  3. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta

    PubMed Central

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has “captured” a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  4. Evolution and organization of the human protein C gene.

    PubMed Central

    Plutzky, J; Hoskins, J A; Long, G L; Crabtree, G R

    1986-01-01

    We have isolated overlapping phage genomic clones covering an area of 21 kilobases that encodes the human protein C gene. The gene is at least 11.2 kilobases long and is made up of nine exons and eight introns. Two regions homologous to epidermal growth factor and transforming growth factor are encoded by amino acids 46-91 and 92-136 and are precisely delimited by introns, as is a similar sequence in the genes for coagulation factor IX and tissue plasminogen activator. When homologous amino acids of factor IX and protein C are aligned, the positions of all eight introns correspond precisely, suggesting that these genes are the product of a relatively recent gene duplication. Nevertheless, the two genes are sufficiently distantly related that no nucleic acid homology remains in the intronic regions and that the size of the introns varies dramatically between the two genes. The similarity of the genes for factor IX and protein C suggests that they may be the most closely related members of the serine protease gene family involved in coagulation and fibrinolysis. Images PMID:3511471

  5. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  6. Rare disease relations through common genes and protein interactions.

    PubMed

    Fernandez-Novo, Sara; Pazos, Florencio; Chagoyen, Monica

    2016-06-01

    ODCs (Orphan Disease Connections), available at http://csbg.cnb.csic.es/odcs, is a novel resource to explore potential molecular relations between rare diseases. These molecular relations have been established through the integration of disease susceptibility genes and human protein-protein interactions. The database currently contains 54,941 relations between 3032 diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. A Highly Conserved gp120 Inner Domain Residue Modulates Env Conformation and Trimer Stability

    PubMed Central

    Ding, Shilei; Tolbert, William D.; Prévost, Jérémie; Pacheco, Beatriz; Coutu, Mathieu; Debbeche, Olfa; Xiang, Shi-Hua

    2016-01-01

    double mutant compared to the wild-type protein but identified higher mobility within the interface between layer 1 and layer 2, the bridging sheet region, and the CD4 binding site. IMPORTANCE HIV-1 Env transitions to the CD4-bound conformation are required for viral entry. Previous studies identified a highly conserved residue of the inner domain, W69, as being involved in these conformational transitions (A. Finzi, S. H. Xiang, B. Pacheco, L. Wang, J. Haight, et al., Mol Cell 37:656–667, 2010, http://dx.doi.org/10.1016/j.molcel.2010.02.012). Here, we show that W69, located at the interface between gp120 and gp41 in the PGT151-bound trimer, plays a critical role in the interprotomer signaling induced by CD4 binding. This new information might be useful in immunogen design. PMID:27384653

  8. SimEnvVis: A Climate Data Visualization Wizard

    NASA Astrophysics Data System (ADS)

    Heitzler, Magnus; Nocke, Thomas

    2013-04-01

    To efficiently make sense of complex climate data, climate scientists need to choose and utilize appropriate analysis tools in respect to specific sets of tasks. Among these, visual analysis tools, like those originating from the field of visual analytics, efficiently support to communicate such information by directly addressing human visual perception. However, climate scientists often are not aware of or not familiar with the large variety of available visual analysis tools or are underestimating their potential benefit for common research tasks and thus reducing the probability to use most suitable ones and therefore impairing the knowledge discovery process. To address this problem, SimEnvVis was developed as an easy-to-use wizard-based software system guiding the user step-by-step in choosing most appropriate visualization and visual analytics tools from a large and easily extendable repository consisting of script-based and interactive tools with different application foci (spatial, temporal or abstract data) and supported techniques (e.g. glyphs, isocontours, stream visualization). Considering the analysis context (e.g. data characteristics, user preferences and analysis tasks) SimEnvVis automatically evaluates the attached tools using a combination of a vector-based and a rule-based mechanism. Based on the users decision, the selected visual analysis tool is launched using a template which is dynamically parameterized by taking into account the analysis context. By displaying the session history in different modes as well as providing the possibility to start SimEnvVis in first-time-user mode to reduce GUI complexity and hide tools which are under development the wizard is in particular useful for novice users. This way, SimEnvVis increases the probability for the usage of appropriate visual analysis tools, lowers the obstacles of familiarization with them and therefore accelerates the knowledge discovery process as well as positively contributes to the

  9. Protein-coding potential of mouse mammary tumor virus genome RNA as examined by in vitro translation.

    PubMed Central

    Dickson, C; Peters, G

    1981-01-01

    The protein-coding capacity of the mouse mammary tumor virus genome has been examined by in vitro translation of genome length and polyadenylated subgenomic fragments of viral RNA. Intact genome RNA of about 35S programmed synthesis of the Pr77gag, Pr110gag and Pr160gag/pol precursors seen in infected cells in vivo. Polyadenylated RNA fragments of 18 to 28S encoded products whose tryptic peptide maps resembled those of the nonglycosylated precursor to the envelope glycoproteins, confirming the gene order 5'-gag-pol-env-3'. Translation of polyadenylated RNA fragments smaller than 18S yielded a series of related proteins whose peptide maps bore no resemblance to any of the virion structural proteins. Thus, a region of the mouse mammary tumor virus genome distal to the env gene appears to have an open reading frame sufficient to encode at least 36,000 daltons of protein as of yet unknown function. Images PMID:6260988

  10. Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

    PubMed Central

    Parikh, Amit; Kumar, Devanand; Chawla, Yogesh; Kurthkoti, Krishna; Khan, Shazia; Varshney, Umesh

    2013-01-01

    Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research. PMID:23315736

  11. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  12. Gene evolution and functions of extracellular matrix proteins in teeth

    PubMed Central

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-01-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  13. Identification of genes involved in radioresistance of nasopharyngeal carcinoma by integrating gene ontology and protein-protein interaction networks.

    PubMed

    Guo, Ya; Zhu, Xiao-Dong; Qu, Song; Li, Ling; Su, Fang; Li, Ye; Huang, Shi-Ting; Li, Dan-Rong

    2012-01-01

    Radioresistance remains one of the important factors in relapse and metastasis of nasopharyngeal carcinoma. Thus, it is imperative to identify genes involved in radioresistance and explore the underlying biological processes in the development of radioresistance. In this study, we used cDNA microarrays to select differential genes between radioresistant CNE-2R and parental CNE-2 cell lines. One hundred and eighty-three significantly differentially expressed genes (p<0.05) were identified, of which 138 genes were upregulated and 45 genes were downregulated in CNE-2R. We further employed publicly available bioinformatics related software, such as GOEAST and STRING to examine the relationship among differentially expressed genes. The results show that these genes were involved in type I interferon-mediated signaling pathway biological processes; the nodes tended to have high connectivity with the EGFR pathway, IFN-related pathways, NF-κB. The node STAT1 has high connectivity with other nodes in the protein-protein interaction (PPI) networks. Finally, the reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and Western blotting. The results were consistent with the microarray data. Our study suggests that microarrays combined with gene ontology and protein interaction networks have great value in the identification of genes of radioresistance in nasopharyngeal carcinoma; genes involved in several biological processes and protein interaction networks may be relevant to NPC radioresistance; in particular, the verified genes CCL5, STAT1-α, STAT2 and GSTP1 may become potential biomarkers for predicting NPC response to radiotherapy.

  14. (Genetic engineering with a gene encoding a soybean storage protein)

    SciTech Connect

    Beachy, R.N.

    1985-12-18

    We have isolated and characterized a gene which encodes the alpha prime subunit of beta conglycinin. This gene was fully sequenced by DNA sequence analysis and a report of that work was prepared and submitted for publication in early November 1985. This represented the culmination of several years of research effort by several scientists. A preprint of that work is attached to this report and has been offered by Dr. J.J. Doyle, Dr. Mary A. Schuler and Dr. Jerry Slighton, as well as myself. This paper is a comparison of the alpha prime subunit gene with a similar gene from phaseolus vulgaris, the common garden bean. In this paper we compare the sequences that are 5' of the gene, and which would represent the transcriptional promoter, as well as the sequences within the structural region of the gene. The sequence paper also compares the amino acid sequence of these two genes with that of other genes from Phaseolus, peas and from soybeans. On the basis of this comparison, we predict evolutionary trends within the multigene families which encode these proteins in the various plants, as well as to look at the protein itself to try to predict regions of the protein that might have functional significance. All of this work was done on a prior DOE-BER grant and has simply been reported here for the first time.

  15. Photosynthesis genes in marine viruses yield proteins during host infection.

    PubMed

    Lindell, Debbie; Jaffe, Jacob D; Johnson, Zackary I; Church, George M; Chisholm, Sallie W

    2005-11-03

    Cyanobacteria, and the viruses (phages) that infect them, are significant contributors to the oceanic 'gene pool'. This pool is dynamic, and the transfer of genetic material between hosts and their phages probably influences the genetic and functional diversity of both. For example, photosynthesis genes of cyanobacterial origin have been found in phages that infect Prochlorococcus and Synechococcus, the numerically dominant phototrophs in ocean ecosystems. These genes include psbA, which encodes the photosystem II core reaction centre protein D1, and high-light-inducible (hli) genes. Here we show that phage psbA and hli genes are expressed during infection of Prochlorococcus and are co-transcribed with essential phage capsid genes, and that the amount of phage D1 protein increases steadily over the infective period. We also show that the expression of host photosynthesis genes declines over the course of infection and that replication of the phage genome is a function of photosynthesis. We thus propose that the phage genes are functional in photosynthesis and that they may be increasing phage fitness by supplementing the host production of these proteins.

  16. Ribosomal protein gene mapping and human chromosomal disorders

    SciTech Connect

    Kenmochi, N.; Goodman, N.; Page, D.C.

    1994-09-01

    In Drosophila, the Minute phenotype (reduced body size, diminished viability and fertility, and short, thin bristles) results from heterozygous deficiencies (deletions) at any one of 50 loci scattered about the genome. A handful of these Minute loci have been molecularly characterized, and all have been found to encode ribosomal proteins. Thus, the Minute phenotype appears to result from reduced protein synthetic capacity in flies with one rather than two copies of a given ribosomal protein (rp) gene. We are pursuing the possibility that similar reductions in protein synthetic capacity--again resulting from rp gene deficiencies--might underlie phenotypes associated with certain chromosomal disorders in humans. We and our colleagues have reported findings consistent with a role for RPS4 deficiency in the etiology of certain features of Turner syndrome, a complex human disorder classically associated with an XO karyotype. We are intrigued by the possibility that deficiencies of other human rp genes might cause phenotypic abnormalities similar to those seen in Turner syndrome--just as deficiencies of any of a number of Drosophila rp genes cause the Minute phenotype. We must first learn the chromosomal map position of each of the estimated 83 human rp genes. The task of mapping the functional (intron-containing) rp genes is complicated by the existence of processed pseudogenes elsewhere in the genome. To date, we have assigned (or confirmed the previous assignment of) 38 rp genes to individual human chromosomes by PCR analysis of human-rodent somatic cell hybrids containing subsets of human chromosomes, with all but four chromosomes carrying at least one rp gene. We have also identified more than 100 large-insert human YAC (yeast artificial chromosome) clones that contain individual rp genes. Such screening of YAC libraries will result in precise positioning of the rp genes on the emerging physical map of the human genome.

  17. Prediction of the Ebola virus infection related human genes using protein-protein interaction network.

    PubMed

    Cao, HuanHuan; Zhang, YuHang; Zhao, Jia; Zhu, Liucun; Wang, Yi; Li, JiaRui; Feng, Yuanming; Zhang, Ning

    2017-03-10

    Ebola hemorrhagic fever (EHF) is caused by Ebola virus (EBOV). It is reported that human could be infected by EBOV with a high fatality rate. However, association factors between EBOV and host still tend to be ambiguous. According to the "guilt by association" (GBA) principle, proteins interacting with each other are very likely to function similarly or the same. Based on this assumption, we tried to obtain EBOV infection-related human genes in a protein-protein interaction network using Dijkstra algorithm. We hope it could contribute to the discovery of novel effective treatments. Finally, 15 genes were selected as potential EBOV infection-related human genes.

  18. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  19. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  20. The p53 gene and protein in human brain tumors

    SciTech Connect

    Louis, D.N. )

    1994-01-01

    Because p53 gene alterations are commonplace in human tumors and because p53 protein is involved in a number of important cellular pathways, p53 has become a topic of intensive investigation, both by basic scientists and clinicians. p53 was initially identified by two independent laboratories in 1979 as a 53 kilodalton (kD) protein that complexes with the large T antigen of SV40 virus. Shortly thereafter, it was shown that the E1B oncoprotein of adenovirus also binds p53. The binding of two different oncogenic viral tumor proteins to the same cellular protein suggested that p53 might be integral to tumorigenesis. The human p53 cDNA and gene were subsequently cloned in the mid-1980s, and analysis of p53 gene alterations in human tumors followed a few year later. During these 10 years, researchers grappling with the vagaries of p53 first characterized the gene as an oncogene, then as a tumor suppressor gene, and most recently as both a tumor suppressor gene and a so-called [open quotes]dominant negative[close quotes] oncogene. The last few years have seen an explosion in work on this single gene and its protein product. A review of a computerized medical database revealed approximately 650 articles on p53 in 1992 alone. p53 has assumed importance in neuro-oncology because p53 mutations and protein alterations are frequent in the common diffuse, fibrillary astrocytic tumors of adults. p53 mutations in astrocytomas were first described in 1989 and were followed by more extensive analyses of gene mutations and protein alterations in adult astrocytomas. The gene has also been studied in less common brain tumors. Elucidating the role of p53 in brain tumorigenesis will not only enhance understanding of brain tumor biology but may also contribute to improved diagnosis and therapy. This discussion reviews key aspects of the p53 gene and protein, and describe their emerging roles in central nervous system neoplasia. 102 refs., 6 figs., 1 tab.

  1. Identification of genes and proteins associated with anagen wool growth.

    PubMed

    Zhao, J; Liu, N; Liu, K; He, J; Yu, J; Bu, R; Cheng, M; De, W; Liu, J; Li, H

    2017-02-01

    Identifying genes of major effect for wool growth would offer strategies for improving the quality and increasing the yield of fine wool. In this study, we employed the Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin (more wool growing) in Aohan fine wool sheep (a Chinese indigenous breed) in comparison with groin skin (no wool growing) at the anagen stage of the wool follicle. A microarray study revealed that 4772 probes were differentially expressed, including 2071 upregulated and 2701 downregulated probes, in the comparisons of body side skin vs. groin skin (S/G). The microarray results were verified by means of quantitative PCR. A total of 1099 probes were assigned to unique genes/transcripts. The number of distinct genes/transcripts (annotated) was 926, of which 352 were upregulated and 574 were downregulated. In S/G, 13 genes were upregulated by more than 10 fold, whereas 60 genes were downregulated by more than 10 fold. Further analysis revealed that the majority of the genes possibly related to the wool growth could be assigned to categories including regulation of cell division, intermediate filament, cytoskeletal part and growth factor activity. Several potential gene families may participate in hair growth regulation, including fibroblast growth factors, transforming growth factor-β, WNTs, insulin-like growth factor, vascular endothelial growth factors and so on. Proteomic analysis also revealed 196 differentially expressed protein points, of which 121 were identified as single protein points.

  2. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  3. The gene-protein database of Escherichia coli: edition 5.

    PubMed

    VanBogelen, R A; Sankar, P; Clark, R L; Bogan, J A; Neidhardt, F C

    1992-12-01

    The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.

  4. Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques.

    PubMed Central

    Overbaugh, J; Rudensey, L M

    1992-01-01

    Genetic diversity is a hallmark of the human immunodeficiency virus (HIV) genome, but the role of distinct HIV variants in the development of AIDS is unclear. Envelope (env) is the most highly variable gene in HIV as well as in other retroviruses. We have previously demonstrated that variation in simian immunodeficiency virus (SIV) env is primarily localized in two regions (V1 and V4) during progression to simian AIDS. To determine whether there is a common genotype that evolves as AIDS develops, a total of 160 SIV env genes isolated directly from the tissue DNAs of four macaques infected with cloned virus were compared. Common amino acid sequence changes were identified within V1, V4, and, in the late stages of disease, near V3. At several positions, the same amino acid change was seen frequently in the variant genomes from all four animals. As AIDS developed, the majority of viruses evolved an extended sequence in V1 that was rich in serine and threonine residues and shared similarity with proteins modified by O-linked glycosylation. Several of the predominant common sequence changes in V1 and V4 created new sites for N-linked glycosylation. Thus, common features of the SIV variants that evolve during progression to AIDS are motifs that potentially allow for structural and functional changes in the env protein as a result of carbohydrate addition. PMID:1527847

  5. Embryonic Expression and Evolution of Duplicated E-Protein Genes in Xenopus Laevis: Parallels with Ancestral E-Protein Genes

    PubMed Central

    Shain, D. H.; Neuman, T.; Zuber, M. X.

    1997-01-01

    E-proteins comprise a subfamily of helix-loop-helix transcription factors that have been identified in arthropods and several chordate taxa. In mammals, there are three classes of E-protein genes (E2A, E2-2, and HEB) that encode related, and often interchangeable, gene products. We have determined that the clawed frog Xenopus laevis contains twice the number of transcriptionally active E-protein genes when compared with other vertebrate species. Based upon genomic Southern blots and nucleotide sequence comparisons, it is likely that the additional X. laevis genes arose from tetraploidization. During embryogenesis, XE2A (homologue of mammalian E2A) transcripts were broadly expressed in anterior and posterior regions of the embryo while homologues of E2-2 (XE2.2) and HEB (XE1.2) appeared in vertebrate-specific structures including the pineal gland, olfactory bulb, and brachial arches. A phylogenetic analysis of these genes and other known metazoan E-proteins suggests that there were two periods of marked E-protein gene expansion; one that predated the radiation of vertebrates, and the other that coincided with Xenopus tetraploidization. Both of these periods were characterized by the rapid evolution of E2-2 and HEB-class genes, but not of E2A. We propose that the former genes acquired new or specialized roles during early chordate evolution and also more recently in Xenopus, as reflected by the stereotypic expression patterns of these genes during X. laevis development. PMID:9136023

  6. Detecting Essential Proteins Based on Network Topology, Gene Expression Data and Gene Ontology Information.

    PubMed

    Zhang, Wei; Xu, Jia; Li, Yuanyuan; Zou, Xiufen

    2016-10-07

    The identification of essential proteins in protein-protein interaction (PPI) networks is of great significance for understanding cellular processes. With the increasing availability of large-scale PPI data, numerous centrality measures based on network topology have been proposed to detect essential proteins from PPI networks. However, most of the current approaches focus mainly on the topological structure of PPI networks, and largely ignore the gene ontology annotation information. In this paper, we propose a novel centrality measure, called TEO, for identifying essential proteins by combining network topology, gene expression profiles and GO information. To evaluate the performance of the TEO method, we compare it with five other methods (degree, betweenness, NC, Pec, CowEWC) in detecting essential proteins from two different yeast PPI datasets. The simulation results show that adding GO information can effectively improve the predicted precision and that our method outperforms the others in predicting essential proteins.

  7. WWOX gene and gene product: tumor suppression through specific protein interactions.

    PubMed

    Salah, Zaidoun; Aqeilan, Rami; Huebner, Kay

    2010-02-01

    The WWOX gene, an archetypal fragile gene, encompasses a chromosomal fragile site at 16q23.2, and encodes the approximately 46-kDa Wwox protein, with WW domains that interact with a growing list of interesting proteins. If the function of a protein is defined by the company it keeps, then Wwox is involved in numerous important signal pathways for bone and germ-cell development, cellular and animal growth and death, transcriptional control and suppression of cancer development. Because alterations to genes at fragile sites are exquisitely sensitive to replication stress-induced DNA damage, there has been an ongoing scientific discussion questioning whether such gene expression alterations provide a selective advantage for clonal expansion of neoplastic cells, and a parallel discussion on why important genes would be present at sites that are susceptible to inactivation. We offer some answers through a description of known WWOX functions.

  8. Gene encoding herbicide safener binding protein

    DOEpatents

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  9. The ribosomal protein genes and Minute loci of Drosophila melanogaster

    PubMed Central

    Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

    2007-01-01

    Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

  10. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

  11. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually “gutted” and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  12. Protein-directed ribosomal frameshifting temporally regulates gene expression

    PubMed Central

    Napthine, Sawsan; Ling, Roger; Finch, Leanne K.; Jones, Joshua D.; Bell, Susanne; Brierley, Ian; Firth, Andrew E.

    2017-01-01

    Programmed −1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins. PMID:28593994

  13. The proteolipid protein gene: Double, double, . . . and trouble

    SciTech Connect

    Hodes, M.E.; Dlouhy, S.R.

    1996-07-01

    That more of a good thing may be too much has been apparent at least since the discovery that Down syndrome is caused by three copies of chromosome 21 instead of the normal two. Duplications of myelin genes also lead to trouble. An extra dose of PMP22, the gene for a protein of peripheral nervous system myelin, causes Charcot-Marie Tooth type 1A disease (CMT1A). Increased dosage of the proteolipid protein gene, PLP, which encodes the chief protein of CNS myelin, can cause Pelizaeus-Merzbacher disease (PMD). The work of Inoue et al. is of particular importance because they found the duplication in four of five families with {open_quotes}classical{close_quotes} PMD, whereas other changes in PLP, such as missense mutations, are found in no more than one in four or five patients with the disease. 27 refs.

  14. NMobTec-EnvEdu: M-Learning System for Environmental Education

    ERIC Educational Resources Information Center

    Cavus, Nadire

    2008-01-01

    This paper introduced the implementation of a New Mobile Technologies and Environmental Education System (NMobTec-EnvEdu) designed for m-learning environments. The NMobTec-EnvEdu system has been developed to provide environmental education in a collaborative framework to undergraduate students through the Internet using mobile phones. The study…

  15. Development and characterization of an equine infectious anemia virus Env-pseudotyped reporter virus.

    PubMed

    Tallmadge, R L; Brindley, M A; Salmans, J; Mealey, R H; Maury, W; Carpenter, S

    2008-07-01

    We developed a replication-defective reporter virus pseudotyped with the envelope glycoprotein of equine infectious anemia virus (EIAV). The in vitro host range and neutralization phenotype of EIAV Env-pseudotyped virus were similar to those of replication-competent virus. An EIAV Env pseudovirus will improve antigenic characterization of viral variants and evaluation of lentivirus vaccines.

  16. NAM-1gene polymorphism and grain protein content in Hordeum.

    PubMed

    Jamar, Catherine; Loffet, Francois; Frettinger, Patrick; Ramsay, Luke; Fauconnier, Marie-Laure; du Jardin, Patrick

    2010-04-15

    Grain protein content (GPC) is a key quality factor for malting and brewing process. In wheat, a QTL explaining a large part of GPC variation was identified, which co-localizes with a gene encoding a NAC transcription factor (TtNAM-B1). NAC transcription factors influence GPC by their role in the regulation of senescence and in protein remobilization. An orthologous gene was discovered on barley chromosome 6H where a GPC QTL was mapped. In this study, we identify allelic variation of the NAM-1 gene for three species of Hordeum representing wild and cultivated barley and we investigate the possible link with GPC. Three haplotypes were identified, one corresponds to the sequences of 11 European varieties representing H. vulgare, one corresponds to the sequence found in H. spontaneum and one represents the sequence of H. bulbosum. Three SNPs were identified between H. spontaneum sequence and H. vulgare sequence. One of the H. bulbosum polymorphisms leads to the introduction of a stop codon and a non-functional protein. Differences in GPC between the 11 varieties were found but no polymorphism in the NAM-1 gene was observed, suggesting that differences in expression of the HvNAM-1 gene or other genes should play a role in GPC regulation. Nevertheless based on published values for GPC of H. bulbosum and H. spontaneum compared to GPC measured here in H. vulgare, the non-functional protein is associated with the lower GPC, suggesting that loss of functionality of the NAM-1 gene in Hordeum is related to lower GPC. Moreover H. spontaneum GPC seems to be higher than H. vulgare GPC, suggesting also that allelic variation of the functional NAM-1 gene could be associated with GPC variation within the genus Hordeum. Copyright 2009 Elsevier GmbH. All rights reserved.

  17. Evolution of yolk protein genes in the Echinodermata.

    PubMed

    Prowse, Thomas A A; Byrne, Maria

    2012-01-01

    Vitellogenin genes (vtg) encode large lipid transfer proteins (LLTPs) that are typically female-specific, functioning as precursors to major yolk proteins (MYPs). Within the phylum Echinodermata, however, the MYP of the Echinozoa (Echinoidea + Holothuroidea) is expressed by an unrelated transferrin-like gene that has a reproductive function in both sexes. We investigated egg proteins in the Asterozoa (Asteroidea + Ophiuroidea), a sister clade to the Echinozoa, showing that eggs of the asteroid Parvulastra exigua contain a vitellogenin protein (Vtg). vtg is expressed by P. exigua, a species with large eggs and nonfeeding larvae, and by the related asterinid Patiriella regularis which has small eggs and feeding larvae. In the Asteroidea, therefore, the reproductive function of vtg is conserved despite significant life history evolution. Like the echinozoan MYP gene, asteroid vtg is expressed in both sexes and may play a role in the development of both ovaries and testes. Phylogenetic analysis indicated that a putative Vtg from the sea urchin genome, a likely pseudogene, does not clade with asteroid Vtg. We propose the following sequence as a potential pathway for the evolution of YP genes in the Echinodermata: (1) the ancestral echinoderm produced YPs derived from Vtg, (2) bisexual vtg expression subsequently evolved in the echinoderm lineage, (3) the reproductive function of vtg was assumed by a transferrin-like gene in the ancestral echinozoan, and (4) redundant echinozoan vtg was released from stabilizing selection.

  18. Identifying Novel Candidate Genes Related to Apoptosis from a Protein-Protein Interaction Network

    PubMed Central

    Wang, Baoman; Yuan, Fei; Kong, Xiangyin; Hu, Lan-Dian; Cai, Yu-Dong

    2015-01-01

    Apoptosis is the process of programmed cell death (PCD) that occurs in multicellular organisms. This process of normal cell death is required to maintain the balance of homeostasis. In addition, some diseases, such as obesity, cancer, and neurodegenerative diseases, can be cured through apoptosis, which produces few side effects. An effective comprehension of the mechanisms underlying apoptosis will be helpful to prevent and treat some diseases. The identification of genes related to apoptosis is essential to uncover its underlying mechanisms. In this study, a computational method was proposed to identify novel candidate genes related to apoptosis. First, protein-protein interaction information was used to construct a weighted graph. Second, a shortest path algorithm was applied to the graph to search for new candidate genes. Finally, the obtained genes were filtered by a permutation test. As a result, 26 genes were obtained, and we discuss their likelihood of being novel apoptosis-related genes by collecting evidence from published literature. PMID:26543496

  19. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2017-03-21

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  20. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  1. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  2. Divinyl ether synthase gene and protein, and uses thereof

    DOEpatents

    Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  3. Gene 5. 5 protein of bacteriophaze T7 inhibits the nucleoid protein H-NS of Escherichia coli

    SciTech Connect

    Liu, Q.; Richardson, C.C. )

    1993-03-01

    Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection. Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps. A fusion protein of gene 5.5 protein and maltose binding protein also purifies with H-NS. The fusion protein binds to the DNA-H-NS complex and abolishes H-NS-mediated inhibition of transcription by Escherichia coli and T7 RNA polymerases in vitro. Expression of gene 5.5 also relieves the repression of the Escherichia coli proU promoter by H-NS in vivo. The change of leucine to proline at residue 30 of gene 5.5 protein abolishes the interaction between gene 5.5 protein and H-NS. 30 refs., 4 figs., 1 tab.

  4. Japanese neuropathy patients with peripheral myelin protein-22 gene aneuploidy

    SciTech Connect

    Lebo, R.V.; Li, L.Y.; Flandermeyer, R.R.

    1994-09-01

    Peripheral myelin protein (PMP-22) gene aneuploidy results in Charcot-Marie-Tooth disease Type 1A (CMT1A) and the Hereditary Neuropathy with Liability to Pressure Palsy (HNPP) in Japanese patients as well as Caucasian Americans. Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, results when expression of one of at least seven genes is defective. CMT1A, about half of all CMT mutations, is usually associated with a duplication spanning the peripheral myelin protein-22 gene on distal chromosome band 17p11.2. Autosomal dominant HNPP (hereditary pressure and sensory neuropathy, HPSN) results from a deletion of the CMT1A gene region. Multicolor in situ hybridization with PMP-22 gene region probe characterized HNPP deletion reliably and detected all different size duplications reported previously. In summary, 72% of 28 Japanese CMT1 (HMSNI) patients tested had the CMT1A duplication, while none of the CMT2 (HMSNII) or CMT3 (HMSNIII) patients had a duplication. Three cases of HNPP were identified by deletion of the CMT1A gene region on chromosome 17p. HNPP and CMT1A have been reported to result simultaneously from the same unequal recombination event. The lower frequency of HNPP compared to CMT1A suggests that HNPP patients have a lower reproductive fitness than CMT1A patients. This result, along with a CMT1A duplication found in an Asian Indian family, demonstrates the broad geographic distribution and high frequency of PMP-22 gene aneuploidy.

  5. Possible eggshell protein gene from Schistosoma mansoni.

    PubMed

    Johnson, K S; Taylor, D W; Cordingley, J S

    1987-01-02

    We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products.

  6. Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

    PubMed Central

    Roy, Nathan H.; Chan, Jany; Lambelé, Marie

    2013-01-01

    HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells. PMID:23637402

  7. Fe-S Proteins that Regulate Gene Expression

    PubMed Central

    Mettert, Erin L.; Kiley, Patricia J.

    2014-01-01

    Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

  8. Challenges in biotechnology at LLNL: from genes to proteins

    SciTech Connect

    Albala, J S

    1999-03-11

    This effort has undertaken the task of developing a link between the genomics, DNA repair and structural biology efforts within the Biology and Biotechnology Research Program at LLNL. Through the advent of the I.M.A.G.E. (Integrated Molecular Analysis of Genomes and their Expression) Consortium, a world-wide effort to catalog the largest public collection of genes, accepted and maintained within BBRP, it is now possible to systematically express the protein complement of these to further elucidate novel gene function and structure. The work has ensued in four phases, outlined as follows: (1) Gene and System selection; (2) Protein expression and purification; (3) Structural analysis; and (4) biological integration. Proteins to be expressed have been those of high programmatic interest. This includes, in particular, proteins involved in the maintenance of genome integrity, particularly those involved in the repair of DNA damage, including ERCC1, ERCC4, XRCC2, XRCC3, XRCC9, HEX1, APN1, p53, RAD51B, RAD51C, and RAD51. Full-length cDNA cognates of selected genes were isolated, and cloned into baculovirus-based expression vectors. The baculoviral expression system for protein over-expression is now well-established in the Albala laboratory. Procedures have been successfully optimized for full-length cDNA clining into expression vectors for protein expression from recombinant constructs. This includes the reagents, cell lines, techniques necessary for expression of recombinant baculoviral constructs in Spodoptera frugiperda (Sf9) cells. The laboratory has also generated a high-throughput baculoviral expression paradigm for large scale expression and purification of human recombinant proteins amenable to automation.

  9. Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation

    PubMed Central

    Martinez, Paola; Sundling, Christopher; O'Dell, Sijy; Mascola, John R.; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

    2015-01-01

    Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines. PMID:25762407

  10. BLyS-mediated modulation of naïve B cell subsets impacts HIV Env-induced antibody responses1

    PubMed Central

    Dosenovic, Pia; Soldemo, Martina; Scholz, Jean L.; O’Dell, Sijy; Grasset, Emilie K.; Pelletier, Nadège; Karlsson, Mikael C. I.; Mascola, John R.; Wyatt, Richard T.; Cancro, Michael P.; Karlsson Hedestam, Gunilla B.

    2012-01-01

    Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs (bNAbs) targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, as the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce bNAbs by vaccination may be due to the use of sub-optimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after antigen encounter. To investigate if perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B Lymphocyte Stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used here substantially affected naïve B cell populations; in particular, it resulted in an increased number of B cells surviving counter-selection at the transitional stages. We also observed an increased number of mature naïve B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab binding titers measured after boosting. The results presented here suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1. PMID:22561155

  11. Expression of heat shock protein genes in insect stress responses

    USDA-ARS?s Scientific Manuscript database

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  12. Linking Protein and RNA Function within the Same Gene.

    PubMed

    Szempruch, Anthony; Guttman, Mitchell

    2017-02-23

    Exposure to ultraviolet light leads to a cell-wide DNA damage response that includes a global reduction in transcription. Williamson et al., identify a protein involved in this process as well as a noncoding RNA produced by alternative processing of RNA transcribed from the same gene that promotes recovery from the repressed state.

  13. Molecular evolution of the mammalian ribosomal protein gene, RPS14.

    PubMed

    Rhoads, D D; Roufa, D J

    1991-07-01

    Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.

  14. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis

    PubMed Central

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-01-01

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the “recycling” of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  15. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance.

  16. Bioengineered Silk Protein-Based Gene Delivery Systems

    PubMed Central

    Numata, Keiji; Subramanian, Balajikarthick; Currie, Heather A.; Kaplan, David L.

    2009-01-01

    Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(l-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30-lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm, showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems. PMID:19577803

  17. Transcriptional regulation of the uncoupling protein-1 gene.

    PubMed

    Villarroya, Francesc; Peyrou, Marion; Giralt, Marta

    2017-03-01

    Regulated transcription of the uncoupling protein-1 (UCP1) gene, and subsequent UCP1 protein synthesis, is a hallmark of the acquisition of the differentiated, thermogenically competent status of brown and beige/brite adipocytes, as well as of the responsiveness of brown and beige/brite adipocytes to adaptive regulation of thermogenic activity. The 5' non-coding region of the UCP1 gene contains regulatory elements that confer tissue specificity, differentiation dependence, and neuro-hormonal regulation to UCP1 gene transcription. Two main regions-a distal enhancer and a proximal promoter region-mediate transcriptional regulation through interactions with a plethora of transcription factors, including nuclear hormone receptors and cAMP-responsive transcription factors. Co-regulators, such as PGC-1α, play a pivotal role in the concerted regulation of UCP1 gene transcription. Multiple interactions of transcription factors and co-regulators at the promoter region of the UCP1 gene result in local chromatin remodeling, leading to activation and increased accessibility of RNA polymerase II and subsequent gene transcription. Moreover, a commonly occurring A-to-G polymorphism in close proximity to the UCP1 gene enhancer influences the extent of UCP1 gene transcription. Notably, it has been reported that specific aspects of obesity and associated metabolic diseases are associated with human population variability at this site. On another front, the unique properties of the UCP1 promoter region have been exploited to develop brown adipose tissue-specific gene delivery tools for experimental purposes. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.

  19. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  20. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  1. Comprehensive functional analysis of large lists of genes and proteins.

    PubMed

    Mlecnik, Bernhard; Galon, Jérôme; Bindea, Gabriela

    2017-03-22

    The interpretation of high dimensional datasets resulting from genomic and proteomic experiments in a timely and efficient manner is challenging. ClueGO software is a Cytoscape App that extracts representative functional biological information for large lists of genes or proteins. The functional enrichment analysis is based on the latest publicly available data from multiple annotation and ontology resources that can be automatically accessed through ClueGO. Predefined settings for the selection of the terms are provided to facilitate the analysis. Results are visualized as networks in which Gene Ontology (GO) terms and pathways are grouped based on their biological role. Many species are now supported by ClueGO and additional organisms are added on demand. ClueGO can be used together with the CluePedia App to enable the visualization of protein-protein interactions within or between pathways.

  2. Sequences of the recA gene and protein.

    PubMed

    Sancar, A; Stachelek, C; Konigsberg, W; Rupp, W D

    1980-05-01

    We have determined the nucleotide sequence of the recA gene of Escherichia coli; this permits the formulation of the primary structure for the recA protein. This structure is consistent with the amino acid composition of the tryptic peptides obtained from the recA protein. The coding region of the recA gene has 1059 base pairs, which specify 352 amino acids. The recA protein has alanine and phenylalanine as its NH2- and COOH-terminal amino acids, respectively, and has the following amino acid composition: Cys3 Asp20 Asn15 Met9 Thr17 Ser20 Glu30 Gln13 Pro10 Gly35 Ala38 Val22 Ile27 Leu31 Tyr7 Phe10 His2Lys27 Trp2 Arg14. Of the three cysteine residues, only two can be alkylated under reducing and denaturing conditions. The molecular weight of the recA polypeptide is 37,842.

  3. Antibodies against Gag are diagnostic markers for feline foamy virus infections while Env and Bet reactivity is undetectable in a substantial fraction of infected cats.

    PubMed

    Romen, Fabian; Pawlita, Michael; Sehr, Peter; Bachmann, Silke; Schröder, Johannes; Lutz, Hans; Löchelt, Martin

    2006-02-20

    Spumaretroviruses or foamy viruses constitute a distinct subfamily of retroviruses. The biology of foamy viruses within the authentic host, their mode of transmission, and disease potential in the authentic host or after zoonotic transmission into human or other species are almost unknown. Using feline foamy virus (FFV) as model system, we established modular enzyme-linked immunosorbent assays (ELISA) suited to determine feline IgG and IgM antibody responses against structural and non-structural FFV proteins. We validated the ELISAs with standard reference sera. In 99 cats admitted to a Swiss veterinary hospital, overall FFV Gag antibody prevalence was 36%, reactivity against Env and the non-structural protein Bet each was about 25%, and 19% of the sera were directed against all three FFV antigens. With one exception, all Bet- and/or Env-positive sera were also positive for Gag. In this small epidemiological pilot study, FFV antibodies were not significantly associated with clinical disease.

  4. SITEX 2.0: Projections of protein functional sites on eukaryotic genes. Extension with orthologous genes.

    PubMed

    Medvedeva, Irina V; Demenkov, Pavel S; Ivanisenko, Vladimir A

    2017-04-01

    Functional sites define the diversity of protein functions and are the central object of research of the structural and functional organization of proteins. The mechanisms underlying protein functional sites emergence and their variability during evolution are distinguished by duplication, shuffling, insertion and deletion of the exons in genes. The study of the correlation between a site structure and exon structure serves as the basis for the in-depth understanding of sites organization. In this regard, the development of programming resources that allow the realization of the mutual projection of exon structure of genes and primary and tertiary structures of encoded proteins is still the actual problem. Previously, we developed the SitEx system that provides information about protein and gene sequences with mapped exon borders and protein functional sites amino acid positions. The database included information on proteins with known 3D structure. However, data with respect to orthologs was not available. Therefore, we added the projection of sites positions to the exon structures of orthologs in SitEx 2.0. We implemented a search through database using site conservation variability and site discontinuity through exon structure. Inclusion of the information on orthologs allowed to expand the possibilities of SitEx usage for solving problems regarding the analysis of the structural and functional organization of proteins. Database URL: http://www-bionet.sscc.ru/sitex/ .

  5. Intron retention in the Drosophila melanogaster Rieske iron sulphur protein gene generated a new protein

    PubMed Central

    Gontijo, Alisson M.; Miguela, Veronica; Whiting, Michael F.; Woodruff, R.C.; Dominguez, Maria

    2011-01-01

    Genomes can encode a variety of proteins with unrelated architectures and activities. It is known that protein-coding genes of de novo origin have significantly contributed to this diversity. However, the molecular mechanisms and evolutionary processes behind these originations are still poorly understood. Here we show that the last 102 codons of a novel gene, Noble, assembled directly from non-coding DNA following an intronic deletion that induced alternative intron retention at the Drosophila melanogaster Rieske Iron Sulphur Protein (RFeSP) locus. A systematic analysis of the evolutionary processes behind the origin of Noble showed that its emergence was strongly biased by natural selection on and around the RFeSP locus. Noble mRNA is shown to encode a bona fide protein that lacks an iron sulphur domain and localizes to mitochondria. Together, these results demonstrate the generation of a novel protein at a naturally selected site. PMID:21610726

  6. Disease Gene Prioritization Based on Topological Similarity in Protein-Protein Interaction Networks

    NASA Astrophysics Data System (ADS)

    Erten, Sinan; Bebek, Gurkan; Koyutürk, Mehmet

    In recent years, many algorithms have been developed to narrow down the set of candidate disease genes implicated by genome wide association studies (GWAS), using knowledge on protein-protein interactions (PPIs). All of these algorithms are based on a common principle; functional association between proteins is correlated with their connectivity/proximity in the PPI network. However, recent research also reveals that networks are organized into recurrent network schemes that underlie the mechanisms of cooperation among proteins with different function, as well as the crosstalk between different cellular processes. In this paper, we hypothesize that proteins that are associated with similar diseases may exhibit patterns of "topological similarity" in PPI networks. Motivated by these observations, we introduce the notion of "topological profile", which represents the location of a protein in the network with respect to other proteins. Based on this notion, we develop a novel measure to assess the topological similarity of proteins in a PPI network. We then use this measure to develop algorithms that prioritize candidate disease genes based on the topological similarity of their products and the products of known disease genes. Systematic experimental studies using an integrated human PPI network and the Online Mendelian Inheritance (OMIM) database show that the proposed algorithm, Vavien, clearly outperforms state-of-the-art network based prioritization algorithms. Vavien is available as a web service at http://www.diseasegenes.org .

  7. The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

    PubMed Central

    Johnson, D G; Carayannopoulos, L; Capra, J D; Tucker, P W; Hanke, J H

    1990-01-01

    All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression. Images PMID:2304473

  8. Gene3D: modelling protein structure, function and evolution.

    PubMed

    Yeats, Corin; Maibaum, Michael; Marsden, Russell; Dibley, Mark; Lee, David; Addou, Sarah; Orengo, Christine A

    2006-01-01

    The Gene3D release 4 database and web portal (http://cathwww.biochem.ucl.ac.uk:8080/Gene3D) provide a combined structural, functional and evolutionary view of the protein world. It is focussed on providing structural annotation for protein sequences without structural representatives--including the complete proteome sets of over 240 different species. The protein sequences have also been clustered into whole-chain families so as to aid functional prediction. The structural annotation is generated using HMM models based on the CATH domain families; CATH is a repository for manually deduced protein domains. Amongst the changes from the last publication are: the addition of over 100 genomes and the UniProt sequence database, domain data from Pfam, metabolic pathway and functional data from COGs, KEGG and GO, and protein-protein interaction data from MINT and BIND. The website has been rebuilt to allow more sophisticated querying and the data returned is presented in a clearer format with greater functionality. Furthermore, all data can be downloaded in a simple XML format, allowing users to carry out complex investigations at their own computers.

  9. Effects of modification of the HIV-1 Env cytoplasmic tail on immunogenicity of VLP vaccines.

    PubMed

    Vzorov, Andrei N; Wang, Li; Chen, Jianjun; Wang, Bao-Zhong; Compans, Richard W

    2016-02-01

    We investigated the effects on assembly and antigenic properties of specific modifications of the transmembrane spanning (TMS) and cytoplasmic tail (CT) domains of HIV-1 Env from a transmitted/founder (T/F) ZM53 Env glycoprotein. A construct containing a short version of the TMS domain derived from the mouse mammary tumor virus (MMTV) Env with or without a GCN4 trimerization sequence in the CT exhibited the highest levels of incorporation into VLPs and induced the highest titers of anti-Env IgG immune responses in a VLP context. Sera from guinea pigs immunized by VLPs with high Env content, and containing the CT trimerization sequence, had increased neutralization activity and antibody avidity. A cross-clade prime-boost regimen with clade B SF162 or clade C ZM53 Env DNA priming and boosting with VLPs containing modified ZM53 Env further enhanced these immune responses. The modified VLPs demonstrate improved potential as HIV-1 vaccine antigens.

  10. Automatic annotation of protein motif function with Gene Ontology terms

    PubMed Central

    Lu, Xinghua; Zhai, Chengxiang; Gopalakrishnan, Vanathi; Buchanan, Bruce G

    2004-01-01

    Background Conserved protein sequence motifs are short stretches of amino acid sequence patterns that potentially encode the function of proteins. Several sequence pattern searching algorithms and programs exist foridentifying candidate protein motifs at the whole genome level. However, amuch needed and importanttask is to determine the functions of the newly identified protein motifs. The Gene Ontology (GO) project is an endeavor to annotate the function of genes or protein sequences with terms from a dynamic, controlled vocabulary and these annotations serve well as a knowledge base. Results This paperpresents methods to mine the GO knowledge base and use the association between the GO terms assigned to a sequence and the motifs matched by the same sequence as evidence for predicting the functions of novel protein motifs automatically. The task of assigning GO terms to protein motifsis viewed as both a binary classification and information retrieval problem, where PROSITE motifs are used as samples for mode training and functional prediction. The mutual information of a motif and aGO term association isfound to be a very useful feature. We take advantageof the known motifs to train a logistic regression classifier, which allows us to combine mutual information with other frequency-based features and obtain a probability of correctassociation. The trained logistic regression model has intuitively meaningful and logically plausible parameter values, and performs very well empirically according to our evaluation criteria. Conclusions In this research, different methods for automatic annotation of protein motifs have been investigated. Empirical result demonstrated that the methods have a great potential for detecting and augmenting information about thefunctions of newly discovered candidate protein motifs. PMID:15345032

  11. Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    PubMed Central

    Pascutti, María Fernanda; Rodríguez, Ana María; Maeto, Cynthia; Perdiguero, Beatriz; Gómez, Carmen E.; Esteban, Mariano; Calamante, Gabriela; Gherardi, María Magdalena

    2012-01-01

    Background Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L). Methodology/Principal Findings BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8+ and CD4+ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8+ T-cells (CD107a/b+) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA's CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens. Conclusions/Significance This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens. PMID:22384183

  12. KEGG as a reference resource for gene and protein annotation

    PubMed Central

    Kanehisa, Minoru; Sato, Yoko; Kawashima, Masayuki; Furumichi, Miho; Tanabe, Mao

    2016-01-01

    KEGG (http://www.kegg.jp/ or http://www.genome.jp/kegg/) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG Orthology (KO) database. The KEGG pathway maps, BRITE hierarchies and KEGG modules are developed as networks of KO nodes, representing high-level functions of the cell and the organism. Currently, more than 4000 complete genomes are annotated with KOs in the KEGG GENES database, which can be used as a reference data set for KO assignment and subsequent reconstruction of KEGG pathways and other molecular networks. As an annotation resource, the following improvements have been made. First, each KO record is re-examined and associated with protein sequence data used in experiments of functional characterization. Second, the GENES database now includes viruses, plasmids, and the addendum category for functionally characterized proteins that are not represented in complete genomes. Third, new automatic annotation servers, BlastKOALA and GhostKOALA, are made available utilizing the non-redundant pangenome data set generated from the GENES database. As a resource for translational bioinformatics, various data sets are created for antimicrobial resistance and drug interaction networks. PMID:26476454

  13. KEGG as a reference resource for gene and protein annotation.

    PubMed

    Kanehisa, Minoru; Sato, Yoko; Kawashima, Masayuki; Furumichi, Miho; Tanabe, Mao

    2016-01-04

    KEGG (http://www.kegg.jp/ or http://www.genome.jp/kegg/) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG Orthology (KO) database. The KEGG pathway maps, BRITE hierarchies and KEGG modules are developed as networks of KO nodes, representing high-level functions of the cell and the organism. Currently, more than 4000 complete genomes are annotated with KOs in the KEGG GENES database, which can be used as a reference data set for KO assignment and subsequent reconstruction of KEGG pathways and other molecular networks. As an annotation resource, the following improvements have been made. First, each KO record is re-examined and associated with protein sequence data used in experiments of functional characterization. Second, the GENES database now includes viruses, plasmids, and the addendum category for functionally characterized proteins that are not represented in complete genomes. Third, new automatic annotation servers, BlastKOALA and GhostKOALA, are made available utilizing the non-redundant pangenome data set generated from the GENES database. As a resource for translational bioinformatics, various data sets are created for antimicrobial resistance and drug interaction networks. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  15. Retroviral display in gene therapy, protein engineering, and vaccine development.

    PubMed

    Urban, Johannes H; Merten, Christoph A

    2011-01-21

    The display and analysis of proteins expressed on biological surfaces has become an attractive tool for the study of molecular interactions in enzymology, protein engineering, and high-throughput screening. Among the growing number of established display systems, retroviruses offer a unique and fully mammalian platform for the expression of correctly folded and post-translationally modified proteins in the context of cell plasma membrane-derived particles. This is of special interest for therapeutic applications such as gene therapy and vaccine development and also offers advantages for the engineering of mammalian proteins toward customized binding affinities and catalytic activities. This review critically summarizes the basic concepts and applications of retroviral display and analyses its benefits in comparison to other display techniques.

  16. Analysis of incomplete gene expression dataset through protein-protein interaction information.

    PubMed

    Massanet-Vila, Raimon; Padró, Teresa; Cardús, Anna; Badimon, Lina; Caminal, Pere; Perera, Alexandre

    2011-01-01

    This paper shows a graph based method to analyze proteomic expression data. The method allows the prediction of the expression of genes not measured by the gene expression technology based on the local connectivity properties of the measured differentially expressed gene set. The prediction of the expression jointly with the stability of this prediction as a function of the variation of the initial expressed set is computed. The method is able to correctly predict one third of the proteins with independence of variations on the selection of the initial set. The algorithm is validated through a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF) protein expression experiment aiming the study of the protein expression patterns and post-translational modifications in human endothelial vascular cells exposed to atherosclerotic levels of Low Density Lipoproteins (LDL).

  17. Structure-Guided Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers

    PubMed Central

    Guenaga, Javier; Dubrovskaya, Viktoriya; de Val, Natalia; Sharma, Shailendra K.; Carrette, Barbara; Ward, Andrew B.

    2015-01-01

    ABSTRACT Due to high viral diversity, an effective HIV-1 vaccine will likely require Envs derived from multiple subtypes to generate broadly neutralizing antibodies (bNAbs). Soluble Env mimics, like the native flexibly linked (NFL) and SOSIP trimers, derived from the subtype A BG505 Env, form homogeneous, stable native-like trimers. However, other Env sequences, such as JRFL and 16055 from subtypes B and C, do so to a lesser degree. The high-resolution BG505 SOSIP crystal structures permit the identification and redesign of Env elements involved in trimer stability. Here, we identified structure trimer-derived (TD) residues that increased the propensity of the subtype B JRFL and subtype C 16055 Env sequences to form well-ordered, homogenous, and highly stable soluble trimers. The generation of these spike mimics no longer required antibody-based selection, positive or negative. Using the redesigned subtype B and C trimer representatives as respective foundations, we further stabilized the NFL TD trimers by engineering an intraprotomer disulfide linkage in the prebridging sheet, I201C-A433C (CC), that locks the gp120 in the receptor nontriggered state. We demonstrated that this disulfide pair prevented CD4 induced-conformational rearrangements in NFL trimers derived from the prototypic subtype A, B, and C representatives. Coupling the TD-based design with the engineered disulfide linkage, CC, increased the propensity of Env to form soluble highly stable spike mimics that are resistant to CD4-induced changes. These advances will allow testing of the hypothesis that such stabilized immunogens will more efficiently elicit neutralizing antibodies in small-animal models and primates. IMPORTANCE HIV-1 displays unprecedented global diversity circulating in the human population. Since the envelope glycoprotein (Env) is the target of neutralizing antibodies, Env-based vaccine candidates that address such diversity are needed. Soluble well-ordered Env mimics, typified by NFL

  18. Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

    PubMed Central

    Porter, D C; Ansardi, D C; Morrow, C D

    1995-01-01

    Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated

  19. Comparative Studies of Vertebrate Beta Integrin Genes and Proteins: Ancient Genes in Vertebrate Evolution

    PubMed Central

    Holmes, Roger S.; Rout, Ujjwal K.

    2011-01-01

    Intregins are heterodimeric α- and β-subunit containing membrane receptor proteins which serve various cell adhesion roles in tissue repair, hemostasis, immune response, embryogenesis and metastasis. At least 18 α- (ITA or ITGA) and 8 β-integrin subunits (ITB or ITGB) are encoded on mammalian genomes. Comparative ITB amino acid sequences and protein structures and ITB gene locations were examined using data from several vertebrate genome projects. Vertebrate ITB genes usually contained 13–16 coding exons and encoded protein subunits with ∼800 amino acids, whereas vertebrate ITB4 genes contained 36-39 coding exons and encoded larger proteins with ∼1800 amino acids. The ITB sequences exhibited several conserved domains including signal peptide, extracellular β-integrin, β-tail domain and integrin β-cytoplasmic domains. Sequence alignments of the integrin β-cytoplasmic domains revealed highly conserved regions possibly for performing essential functions and its maintenance during vertebrate evolution. With the exception of the human ITB8 sequence, the other ITB sequences shared a predicted 19 residue α-helix for this region. Potential sites for regulating human ITB gene expression were identified which included CpG islands, transcription factor binding sites and microRNA binding sites within the 3′-UTR of human ITB genes. Phylogenetic analyses examined the relationships of vertebrate beta-integrin genes which were consistent with four major groups: 1: ITB1, ITB2, ITB7; 2: ITB3, ITB5, ITB6; 3: ITB4; and 4: ITB8 and a common evolutionary origin from an ancestral gene, prior to the appearance of fish during vertebrate evolution. The phylogenetic analyses revealed that ITB4 is the most likely primordial form of the vertebrate β integrin subunit encoding genes, that is the only β subunit expressed as a constituent of the sole integrin receptor ‘α6β4’ in the hemidesmosomes of unicellular organisms. PMID:24970121

  20. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html.

  1. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed Central

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html. PMID:8594596

  2. An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the env precursor interferes with membrane fusion at a post-CD4 binding step.

    PubMed

    Barbouche, R; Decroly, E; Kieny, M P; Fenouillet, E

    2000-07-20

    CLIV is a multiple antigen peptide ([PTKAKRRVVQREKR](4)-K(2)-K-betaA) that encompasses the cleavage region of the human immunodeficiency virus type 1 (HIV-1) envelope precursor. It displays an antiviral activity against HIV-1 and HIV-2 and inhibits HIV-1 Env-mediated cell-to-cell fusion. This effect has previously been attributed to interference with Env processing, resulting in the expression of a nonfusogenic envelope [Virology (1998) 247, 137]. However, we show here that CLIV does not alter the status of Env cleavage at steady state. Using various aggregation/syncytium assays that allow us to discriminate between gp120/CD4 binding and binding followed by gp41-mediated fusion, we demonstrate that CLIV inhibits a step of the cell-to-cell fusion process after CD4 binding. We demonstrate also that CLIV binds at 37 degrees C to a single class of protein present at the CD4(+) cell surface (Scatchard analysis: K(d) = 8 nM; B(max) = 10(4) sites/cell) and that the fusion inhibition activity seems to correlate with binding to this proteic component. In contrast, CLIV interacts with neither membrane-inserted nor CD4-associated Env. We therefore propose that CLIV interferes after Env/CD4 binding with a step of the membrane fusion process that may involve the C-terminal domain of gp120. Copyright 2000 Academic Press.

  3. RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

    DTIC Science & Technology

    2010-06-01

    Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein . J Biol Chem 1996, 271(14):8144-8151. 21. Meisner NC, Hackermuller J...Hauptmann S: Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast...SH3 domain, ankyrin repeat and pH domain 3 tumor microarray reveals 47 annotated genes up regulated in the HA-HuR overexpressing tumors as compared to

  4. An improved method for scoring protein-protein interactions using semantic similarity within the gene ontology.

    PubMed

    Jain, Shobhit; Bader, Gary D

    2010-11-15

    Semantic similarity measures are useful to assess the physiological relevance of protein-protein interactions (PPIs). They quantify similarity between proteins based on their function using annotation systems like the Gene Ontology (GO). Proteins that interact in the cell are likely to be in similar locations or involved in similar biological processes compared to proteins that do not interact. Thus the more semantically similar the gene function annotations are among the interacting proteins, more likely the interaction is physiologically relevant. However, most semantic similarity measures used for PPI confidence assessment do not consider the unequal depth of term hierarchies in different classes of cellular location, molecular function, and biological process ontologies of GO and thus may over-or under-estimate similarity. We describe an improved algorithm, Topological Clustering Semantic Similarity (TCSS), to compute semantic similarity between GO terms annotated to proteins in interaction datasets. Our algorithm, considers unequal depth of biological knowledge representation in different branches of the GO graph. The central idea is to divide the GO graph into sub-graphs and score PPIs higher if participating proteins belong to the same sub-graph as compared to if they belong to different sub-graphs. The TCSS algorithm performs better than other semantic similarity measurement techniques that we evaluated in terms of their performance on distinguishing true from false protein interactions, and correlation with gene expression and protein families. We show an average improvement of 4.6 times the F1 score over Resnik, the next best method, on our Saccharomyces cerevisiae PPI dataset and 2 times on our Homo sapiens PPI dataset using cellular component, biological process and molecular function GO annotations.

  5. An improved method for scoring protein-protein interactions using semantic similarity within the gene ontology

    PubMed Central

    2010-01-01

    Background Semantic similarity measures are useful to assess the physiological relevance of protein-protein interactions (PPIs). They quantify similarity between proteins based on their function using annotation systems like the Gene Ontology (GO). Proteins that interact in the cell are likely to be in similar locations or involved in similar biological processes compared to proteins that do not interact. Thus the more semantically similar the gene function annotations are among the interacting proteins, more likely the interaction is physiologically relevant. However, most semantic similarity measures used for PPI confidence assessment do not consider the unequal depth of term hierarchies in different classes of cellular location, molecular function, and biological process ontologies of GO and thus may over-or under-estimate similarity. Results We describe an improved algorithm, Topological Clustering Semantic Similarity (TCSS), to compute semantic similarity between GO terms annotated to proteins in interaction datasets. Our algorithm, considers unequal depth of biological knowledge representation in different branches of the GO graph. The central idea is to divide the GO graph into sub-graphs and score PPIs higher if participating proteins belong to the same sub-graph as compared to if they belong to different sub-graphs. Conclusions The TCSS algorithm performs better than other semantic similarity measurement techniques that we evaluated in terms of their performance on distinguishing true from false protein interactions, and correlation with gene expression and protein families. We show an average improvement of 4.6 times the F1 score over Resnik, the next best method, on our Saccharomyces cerevisiae PPI dataset and 2 times on our Homo sapiens PPI dataset using cellular component, biological process and molecular function GO annotations. PMID:21078182

  6. PPISEARCHENGINE: gene ontology-based search for protein-protein interactions.

    PubMed

    Park, Byungkyu; Cui, Guangyu; Lee, Hyunjin; Huang, De-Shuang; Han, Kyungsook

    2013-01-01

    This paper presents a new search engine called PPISearchEngine which finds protein-protein interactions (PPIs) using the gene ontology (GO) and the biological relations of proteins. For efficient retrieval of PPIs, each GO term is assigned a prime number and the relation between the terms is represented by the product of prime numbers. This representation is hidden from users but facilitates the search for the interactions of a query protein by unique prime factorisation of the number that represents the query protein. For a query protein, PPISearchEngine considers not only the GO term associated with the query protein but also the GO terms at the lower level than the GO term in the GO hierarchy, and finds all the interactions of the query protein which satisfy the search condition. In contrast, the standard keyword-matching or ID-matching search method cannot find the interactions of a protein unless the interactions involve a protein with explicit annotations. To the best of our knowledge, this search engine is the first method that can process queries like 'for protein p with GO [Formula: see text], find p's interaction partners with GO [Formula: see text]'. PPISearchEngine is freely available to academics at http://search.hpid.org/.

  7. Controlling for Gene Expression Changes in Transcription Factor Protein Networks*

    PubMed Central

    Banks, Charles A. S.; Lee, Zachary T.; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D.; Wen, Zhihui; Hattem, Gaye L.; Seidel, Chris W.; Florens, Laurence; Washburn, Michael P.

    2014-01-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein–protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. PMID:24722732

  8. CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef.

    PubMed Central

    Chen, B K; Gandhi, R T; Baltimore, D

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) genes vpu, env, and nef have all been implicated in modulating the levels of cell surface CD4 on infected cells. To quantitatively assess the relative contribution of each gene product to the regulation of CD4 during HIV infection of Jurkat T cells and peripheral blood mononuclear cells, we have developed an infectious HIV reporter system which expresses different combinations of these genes. To distinguish infected cells in the early or late stages of infection from uninfected cells, these viruses were designed to express human placental alkaline phosphatase with the kinetics of either early or late viral genes. Flow cytometry to detect placental alkaline phosphatase and CD4 in infected cells showed that vpu, env, and nef are independently capable of down-modulation of CD4. As predicted by their respective expression patterns, nef down-modulated CD4 rapidly during the early phase of virus infection whereas vpu and env functioned late in the infection. In both Jurkat cells and peripheral blood mononuclear cells, a combination of the three genes was more efficient than any one or two genes, demonstrating that all three genes are required to achieve maximal CD4 down-modulation. In primary cells, down-modulation of CD4 was less efficient than in Jurkat cells and there was a stronger dependence on nef function for reducing cell surface CD4. HIV therefore has three genes that are able to independently down-modulate CD4; together, they can eliminate the bulk of cell surface CD4. PMID:8709227

  9. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  10. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  11. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    PubMed

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  12. Evolution of Drosophila ribosomal protein gene core promoters

    PubMed Central

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2011-01-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module. PMID:19059316

  13. Evolution of Drosophila ribosomal protein gene core promoters.

    PubMed

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  14. Rational design of orthogonal libraries of protein coding genes.

    PubMed

    Ryan, Daniel; Papamichail, Dimitris

    2013-05-17

    Array-based oligonucleotide synthesis technologies provide access to thousands of custom-designed sequence variants at low cost. Large-scale synthesis and high-throughput assays have become valuable experimental tools to study in detail the interplay between sequence and function. We have developed a methodology and corresponding algorithms for the design of diverse protein coding gene libraries, to exploit the potential of multiplex synthesis and help elucidate the effects of codon utilization and other factors in gene expression. Using our algorithm, we have computationally designed gene libraries with hundreds to thousands of orthogonal codon usage variants, uniformly exploring the design space of codon utilization, while demanding only a small fraction of the synthesis cost that would be required if these variants were synthesized independently.

  15. Prolactin receptor and signal transduction to milk protein genes

    SciTech Connect

    Djiane, J.; Daniel, N.; Bignon, C.

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  16. Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.

    PubMed

    Simmons, Jason; D'Souza, Olivia; Rheault, Mark; Donly, Cam

    2013-02-01

    Many insect species exhibit pesticide-resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real-time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.

  17. Protein-protein interaction inference based on semantic similarity of Gene Ontology terms.

    PubMed

    Zhang, Shu-Bo; Tang, Qiang-Rong

    2016-07-21

    Identifying protein-protein interactions is important in molecular biology. Experimental methods to this issue have their limitations, and computational approaches have attracted more and more attentions from the biological community. The semantic similarity derived from the Gene Ontology (GO) annotation has been regarded as one of the most powerful indicators for protein interaction. However, conventional methods based on GO similarity fail to take advantage of the specificity of GO terms in the ontology graph. We proposed a GO-based method to predict protein-protein interaction by integrating different kinds of similarity measures derived from the intrinsic structure of GO graph. We extended five existing methods to derive the semantic similarity measures from the descending part of two GO terms in the GO graph, then adopted a feature integration strategy to combines both the ascending and the descending similarity scores derived from the three sub-ontologies to construct various kinds of features to characterize each protein pair. Support vector machines (SVM) were employed as discriminate classifiers, and five-fold cross validation experiments were conducted on both human and yeast protein-protein interaction datasets to evaluate the performance of different kinds of integrated features, the experimental results suggest the best performance of the feature that combines information from both the ascending and the descending parts of the three ontologies. Our method is appealing for effective prediction of protein-protein interaction.

  18. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  19. Interferon-stimulated gene 15 and the protein ISGylation system.

    PubMed

    Zhang, Dongxian; Zhang, Dong-Er

    2011-01-01

    Interferon-stimulated gene 15 (ISG15) is one of the most upregulated genes upon Type I interferon treatment or pathogen infection. Its 17  kDa protein product, ISG15, was the first ubiquitin-like modifier identified, and is similar to a ubiquitin linear dimer. As ISG15 modifies proteins in a similar manner to ubiquitylation, protein conjugation by ISG15 is termed ISGylation. Some of the primary enzymes that promote ISGylation are also involved in ubiquitin conjugation. The process to remove ISG15 from its conjugated proteins, termed de-ISGylation, is performed by a cellular ISG15-specific protease, ubiquitin-specific proteases with molecular mass 43 kDa (UBP43)/ubiquitin-specific proteases 18. Relative to ubiquitin, the biological function of ISG15 is still poorly understood, but ISG15 appears to play important roles in various biological and cellular functions. Therefore, there is growing interest in ISG15, as the study of free ISG15 and functional consequences of ISGylation/de-ISGylation may identify useful therapeutic targets. This review highlights recent discoveries and remaining questions important to understanding the biological functions of ISG15.

  20. Analysis of the prion protein gene in multiple system atrophy.

    PubMed

    Chelban, Viorica; Manole, Andreea; Pihlstrøm, Lasse; Schottlaender, Lucia; Efthymiou, Stephanie; OConnor, Emer; Meissner, Wassilios G; Holton, Janice L; Houlden, Henry

    2017-01-01

    Neurodegenerative diseases are a very diverse group of disorders but they share some common mechanisms such as abnormally misfolded proteins with prion-like propagation and aggregation. Creutzfeldt-Jakob disease (CJD) is the most prevalent prion disease in humans. In the sporadic form of CJD the only known risk factor is the codon 129 polymorphism. Recent reports suggested that α-synuclein in multiple system atrophy (MSA) has similar pathogenic mechanisms as the prion protein. Here we present 1 Italian family with MSA and prion disease. Also, cases of concurrent MSA and prion pathology in the same individual or family suggest the possibility of molecular interaction between prion protein and α-synuclein in the process of protein accumulation and neurodegeneration, warranting further investigations. We assessed the PRNP gene by whole-exome sequencing in 264 pathologically confirmed MSA cases and 462 healthy controls to determine whether the 2 diseases share similar risk factors. We then analyzed codon 129 polymorphism by Sanger sequencing and compared with previously published results in sporadic CJD. Homozygosity at codon 129 was present in 50% of pathologically confirmed MSA cases and in 58% of normal controls (odds ratio, 0.7 (95% confidence interval of 0.5-0.9)) compared with 88.2% in sporadic CJD. Our data show that the homozygous state of position 129 in the PRNP is not a risk factor for MSA. No other variants in the PRNP gene were associated with increased risk for MSA.

  1. Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer

    PubMed Central

    2016-01-01

    Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. PMID:27992602

  2. Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer.

    PubMed

    Khasnis, Mukta D; Halkidis, Konstantine; Bhardwaj, Anshul; Root, Michael J

    2016-12-01

    Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI.

  3. Phylogenetics of HIV-1 subtype G env: Greater complexity and older origins than previously reported.

    PubMed

    Tongo, Marcel; Essomba, René G; Nindo, Frederick; Abrahams, Fatima; Nanfack, Aubin Joseph; Fokam, Joseph; Takou, Desire; Torimiro, Judith N; Mpoudi-Ngole, Eitel; Burgers, Wendy A; Martin, Darren P; Dorfman, Jeffrey R

    2015-10-01

    HIV-1 subtype G has played an early and central role in the emergent complexity of the HIV-1 group M (HIV-1M) epidemic in central/west Africa. Here, we analysed new subtype G env sequences sampled from 8 individuals in Yaoundé, Cameroon during 2007-2010, together with all publically available subtype G-attributed full-length env sequences with known sampling dates and locations. We inferred that the most recent common ancestor (MRCA) of the analysed subtype G env sequences most likely occurred in ∼1953 (95% Highest Posterior Density interval [HPD] 1939-1963): about 15 years earlier than previous estimates. We found that the subtype G env phylogeny has a complex structure including seven distinct lineages, each likely dating back to the late 1960s or early 1970s. Sequences from Angola, Gabon and the Democratic Republic of Congo failed to group consistently in these lineages, possibly because they are related to more ancient sequences that are poorly sampled. The circulating recombinant form (CRF), CRF06_cpx env sequences but not CRF25_cpx env sequences are phylogenetically nested within the subtype G clade. This confirms that the CRF06_cpx env plausibly was derived through recombination from a subtype G parent, and suggests that the CRF25_cpx env was likely derived from an HIV-1M lineage related to the MRCA of subtype G that has remained undiscovered and may be extinct. Overall, this fills important gaps in our knowledge of the early events in the spread of HIV-1M.

  4. Next Generation Delivery System for Proteins and Genes of Therapeutic Purpose: Why and How?

    PubMed Central

    Priya Doss, C. George; Lee, Sang-Soo

    2014-01-01

    Proteins and genes of therapeutic interests in conjunction with different delivery systems are growing towards new heights. “Next generation delivery systems” may provide more efficient platform for delivery of proteins and genes. In the present review, snapshots about the benefits of proteins or gene therapy, general procedures for therapeutic protein or gene delivery system, and different next generation delivery system such as liposome, PEGylation, HESylation, and nanoparticle based delivery have been depicted with their detailed explanation. PMID:25126554

  5. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    NASA Technical Reports Server (NTRS)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  6. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    NASA Technical Reports Server (NTRS)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  7. Genes encoding calmodulin-binding proteins in the Arabidopsis genome.

    PubMed

    Reddy, Vaka S; Ali, Gul S; Reddy, Anireddy S N

    2002-03-22

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  8. Molecular evolution of monotreme and marsupial whey acidic protein genes.

    PubMed

    Sharp, Julie A; Lefèvre, Christophe; Nicholas, Kevin R

    2007-01-01

    Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

  9. Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail

    PubMed Central

    Muranyi, Walter; Malkusch, Sebastian; Müller, Barbara; Heilemann, Mike; Kräusslich, Hans-Georg

    2013-01-01

    The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse. PMID:23468635

  10. Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes.

    PubMed

    Dodds, Peter N; Lawrence, Gregory J; Catanzariti, Ann-Maree; Teh, Trazel; Wang, Ching-I A; Ayliffe, Michael A; Kobe, Bostjan; Ellis, Jeffrey G

    2006-06-06

    Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R-Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R-Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R-Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant-pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.

  11. Diverse antibody genetic and recognition properties revealed following HIV-1 Env immunization

    PubMed Central

    Phad, Ganesh E.; Bernat, Néstor Vázquez; Feng, Yu; Ingale, Jidnyasa; Murillo, Paola Andrea Martinez; O’Dell, Sijy; Li, Yuxing; Mascola, John R.; Sundling, Christopher; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

    2015-01-01

    Isolation of monoclonal antibodies (MAbs) elicited by vaccination provides opportunities to define the development of effective immunity. Ab responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens display narrow neutralizing activity with limited capacity to block infection by tier 2 viruses. Intense work in the field suggests that improved Env immunogens are forthcoming and it is therefore important to concurrently develop approaches to investigate the quality of vaccine-elicited responses at a higher level of resolution. Here, we cloned a representative set of MAbs elicited by a model Env immunogen in rhesus macaques and comprehensively characterized their genetic and functional properties. The MAbs were genetically diverse, even within groups of Abs targeting the same sub-region of Env, consistent with a highly polyclonal response. MAbs directed against two sub-determinants of Env, the CD4 binding site (CD4bs) and the V3 region, could in part account for the neutralizing activity observed in the plasma of the animal from which they were cloned, demonstrating the power of MAb isolation for a detailed understanding of the elicited response. Finally, through comparative analyses of MAb binding and neutralizing capacity of HIV-1 using matched Envs, we demonstrate complex relationships between epitope recognition and accessibility, highlighting the protective quaternary packing of the HIV-1 spike relative to vaccine-induced MAbs. PMID:25964491

  12. N-terminally myristoylated feline foamy virus Gag allows Env-independent budding of sub-viral particles.

    PubMed

    Liu, Yang; Kim, Yong-Boum; Löchelt, Martin

    2011-11-01

    Foamy viruses (FVs) are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP) release. In primate and feline FVs (PFV and FFV), particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal) overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N-terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation.

  13. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  14. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.

    PubMed

    Blackburn, Matthew C; Petrova, Ekaterina; Correia, Bruno E; Maerkl, Sebastian J

    2016-04-20

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

  15. Unusual Fusion Proteins of HIV-1

    PubMed Central

    Langer, Simon; Sauter, Daniel

    2017-01-01

    Despite its small genome size, the Human Immunodeficiency Virus 1 (HIV-1) is one of the most successful pathogens and has infected more than 70 million people worldwide within the last decades. In total, HIV-1 expresses 16 canonical proteins from only nine genes within its 10 kb genome. Expression of the structural genes gag, pol, and env, the regulatory genes rev and tat and the accessory genes vpu, nef, vpr, and vif enables assembly of the viral particle, regulates viral gene transcription, and equips the virus to evade or counteract host immune responses. In addition to the canonically expressed proteins, a growing number of publications describe the existence of non-canonical fusion proteins in HIV-1 infected cells. Most of them are encoded by the tat-env-rev locus. While the majority of these fusion proteins (e.g., TNV/p28tev, p186Drev, Tat1-Rev2, Tat^8c, p17tev, or Ref) are the result of alternative splicing events, Tat-T/Vpt is produced upon programmed ribosomal frameshifting, and a Rev1-Vpu fusion protein is expressed due to a nucleotide polymorphism that is unique to certain HIV-1 clade A and C strains. A better understanding of the expression and activity of these non-canonical viral proteins will help to dissect their potential role in viral replication and reveal how HIV-1 optimized the coding potential of its genes. The goal of this review is to provide an overview of previously described HIV-1 fusion proteins and to summarize our current knowledge of their expression patterns and putative functions. PMID:28119676

  16. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    PubMed

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity.

  17. Prioritization of candidate genes for cattle reproductive traits, based on protein-protein interactions, gene expression, and text-mining.

    PubMed

    Hulsegge, Ina; Woelders, Henri; Smits, Mari; Schokker, Dirkjan; Jiang, Li; Sørensen, Peter

    2013-05-15

    Reproduction is of significant economic importance in dairy cattle. Improved understanding of mechanisms that control estrous behavior and other reproduction traits could help in developing strategies to improve and/or monitor these traits. The objective of this study was to predict and rank genes and processes in brain areas and pituitary involved in reproductive traits in cattle using information derived from three different data sources: gene expression, protein-protein interactions, and literature. We identified 59, 89, 53, 23, and 71 genes in bovine amygdala, dorsal hypothalamus, hippocampus, pituitary, and ventral hypothalamus, respectively, potentially involved in processes underlying estrus and estrous behavior. Functional annotation of the candidate genes points to a number of tissue-specific processes of which the "neurotransmitter/ion channel/synapse" process in the amygdala, "steroid hormone receptor activity/ion binding" in the pituitary, "extracellular region" in the ventral hypothalamus, and "positive regulation of transcription/metabolic process" in the dorsal hypothalamus are most prominent. The regulation of the functional processes in the various tissues operate at different biological levels, including transcriptional, posttranscriptional, extracellular, and intercellular signaling levels.

  18. Gene Ontology Function prediction in Mollicutes using Protein-Protein Association Networks

    PubMed Central

    2011-01-01

    Background Many complex systems can be represented and analysed as networks. The recent availability of large-scale datasets, has made it possible to elucidate some of the organisational principles and rules that govern their function, robustness and evolution. However, one of the main limitations in using protein-protein interactions for function prediction is the availability of interaction data, especially for Mollicutes. If we could harness predicted interactions, such as those from a Protein-Protein Association Networks (PPAN), combining several protein-protein network function-inference methods with semantic similarity calculations, the use of protein-protein interactions for functional inference in this species would become more potentially useful. Results In this work we show that using PPAN data combined with other approximations, such as functional module detection, orthology exploitation methods and Gene Ontology (GO)-based information measures helps to predict protein function in Mycoplasma genitalium. Conclusions To our knowledge, the proposed method is the first that combines functional module detection among species, exploiting an orthology procedure and using information theory-based GO semantic similarity in PPAN of the Mycoplasma species. The results of an evaluation show a higher recall than previously reported methods that focused on only one organism network. PMID:21486441

  19. p53 gene alterations and protein accumulation in colorectal cancer

    PubMed Central

    Bertorelle, R; Esposito, G; Belluco, C; Bonaldi, L; Del Mistro, A; Nitti, D; Lise, M; Chieco-Bianchi, L

    1996-01-01

    Aim—To correlate immunohistochemical staining with single strand conformation polymorphism (SSCP) analysis of the p53 gene in colorectal cancer in order to understand how the findings provided by the two techniques complement each other in defining p53 functional status. Methods—Frozen tumour tissue from 94 patients with colorectal cancer was studied for p53 protein accumulation and gene mutations. Accumulation of p53 protein was detected by immunohistochemistry using PAb1801 and BP53-12-1 monoclonal antibodies. The findings were then compared with SSCP analysis of exons 5 to 8 of the p53 gene. All cases with a positive result by SSCP analysis were confirmed by sequencing. Results—Nuclear staining was observed in 51 (54.2%) cases. SSCP analysis of the DNA amplified by PCR revealed that the electrophoretic pattern had shifted in 30 cases; sequence analysis confirmed the occurrence of a mutation in 29 cases and of a polymorphism in one. In 27 cases both assays gave a positive result, and in 40 both were negative; therefore, concordance between PCR-SSCP and immunohistochemistry was seen in 72% of cases. Conclusion—The data indicate that positive immunostaining corresponds with the presence of a mutation in most, but not all, cases studied; other mechanisms could be responsible for stabilisation and accumulation of p53 protein in the nucleus. Nonsense mutations which do not confer stability on the protein will not be detected by immunohistochemistry and false negative results can also occur with SSCP analysis. Images PMID:16696056

  20. Diverse nucleotide compositions and sequence fluctuation in Rubisco protein genes

    NASA Astrophysics Data System (ADS)

    Holden, Todd; Dehipawala, S.; Cheung, E.; Bienaime, R.; Ye, J.; Tremberger, G., Jr.; Schneider, P.; Lieberman, D.; Cheung, T.

    2011-10-01

    The Rubisco protein-enzyme is arguably the most abundance protein on Earth. The biology dogma of transcription and translation necessitates the study of the Rubisco genes and Rubisco-like genes in various species. Stronger correlation of fractal dimension of the atomic number fluctuation along a DNA sequence with Shannon entropy has been observed in the studied Rubisco-like gene sequences, suggesting a more diverse evolutionary pressure and constraints in the Rubisco sequences. The strategy of using metal for structural stabilization appears to be an ancient mechanism, with data from the porphobilinogen deaminase gene in Capsaspora owczarzaki and Monosiga brevicollis. Using the chi-square distance probability, our analysis supports the conjecture that the more ancient Rubisco-like sequence in Microcystis aeruginosa would have experienced very different evolutionary pressure and bio-chemical constraint as compared to Bordetella bronchiseptica, the two microbes occupying either end of the correlation graph. Our exploratory study would indicate that high fractal dimension Rubisco sequence would support high carbon dioxide rate via the Michaelis- Menten coefficient; with implication for the control of the whooping cough pathogen Bordetella bronchiseptica, a microbe containing a high fractal dimension Rubisco-like sequence (2.07). Using the internal comparison of chi-square distance probability for 16S rRNA (~ E-22) versus radiation repair Rec-A gene (~ E-05) in high GC content Deinococcus radiodurans, our analysis supports the conjecture that high GC content microbes containing Rubisco-like sequence are likely to include an extra-terrestrial origin, relative to Deinococcus radiodurans. Similar photosynthesis process that could utilize host star radiation would not compete with radiation resistant process from the biology dogma perspective in environments such as Mars and exoplanets.

  1. Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

    PubMed Central

    Larkin, J C; Woolford, J L

    1983-01-01

    Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed. Images PMID:6338478

  2. Leber congenital amaurosis: genes, proteins and disease mechanisms.

    PubMed

    den Hollander, Anneke I; Roepman, Ronald; Koenekoop, Robert K; Cremers, Frans P M

    2008-07-01

    Leber congenital amaurosis (LCA) is the most severe retinal dystrophy causing blindness or severe visual impairment before the age of 1 year. Linkage analysis, homozygosity mapping and candidate gene analysis facilitated the identification of 14 genes mutated in patients with LCA and juvenile retinal degeneration, which together explain approximately 70% of the cases. Several of these genes have also been implicated in other non-syndromic or syndromic retinal diseases, such as retinitis pigmentosa and Joubert syndrome, respectively. CEP290 (15%), GUCY2D (12%), and CRB1 (10%) are the most frequently mutated LCA genes; one intronic CEP290 mutation (p.Cys998X) is found in approximately 20% of all LCA patients from north-western Europe, although this frequency is lower in other populations. Despite the large degree of genetic and allelic heterogeneity, it is possible to identify the causative mutations in approximately 55% of LCA patients by employing a microarray-based, allele-specific primer extension analysis of all known DNA variants. The LCA genes encode proteins with a wide variety of retinal functions, such as photoreceptor morphogenesis (CRB1, CRX), phototransduction (AIPL1, GUCY2D), vitamin A cycling (LRAT, RDH12, RPE65), guanine synthesis (IMPDH1), and outer segment phagocytosis (MERTK). Recently, several defects were identified that are likely to affect intra-photoreceptor ciliary transport processes (CEP290, LCA5, RPGRIP1, TULP1). As the eye represents an accessible and immune-privileged organ, it appears to be uniquely suitable for human gene replacement therapy. Rodent (Crb1, Lrat, Mertk, Rpe65, Rpgrip1), avian (Gucy2D) and canine (Rpe65) models for LCA and profound visual impairment have been successfully corrected employing adeno-associated virus or lentivirus-based gene therapy. Moreover, phase 1 clinical trials have been carried out in humans with RPE65 deficiencies. Apart from ethical considerations inherently linked to treating children, major

  3. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  4. Transcription of antifreeze protein genes in Choristoneura fumiferana.

    PubMed

    Qin, W; Doucet, D; Tyshenko, M G; Walker, V K

    2007-08-01

    Antifreeze proteins (AFPs) are encoded by approximately 17 genes in the spruce budworm, Choristoneura fumiferana. Northern analysis using 6 different cDNA probes showed isoform-specific patterns that varied during development. Transcripts for the majority of isoforms were most abundant in the second instar overwintering stage, but some were also detected in first instar and even in egg stages. In situ hybridization using riboprobes corresponding to two 9 kDa protein isoforms showed differential AFP expression even in second instars; CfAFP10 RNA was detected in all tissues, but CfAFP337 RNA distribution was more limited. Two genomic regions encoding three AFP genes have been isolated. Presumptive regulatory regions conferred transcriptional activity when placed upstream of a luciferase reporter sequence and transfected into a C. fumiferana cell line. The CfAFP2.26 core promoter is an 87 bp sequence containing a TATA box, whereas the CfAFP2.7 core promoter is a 76 bp sequence with both a TATA box and CAAT box, which directed higher reporter activities when tested in vitro. Reporter activity was not enhanced with five different hormones, although lower activities were observed with all intron-containing constructs. AFP message half-life, as assessed using reporter assays, was not appreciably influenced by isoform-specific-3'UTRs. These studies successfully demonstrate the temporal and spatial diversity of AFP expression encoded by this small gene family, and underscore the complexity of their regulation.

  5. Protein-Protein Interactions: Gene Acronym Redundancies and Current Limitations Precluding Automated Data Integration.

    PubMed

    Casado-Vela, Juan; Matthiesen, Rune; Sellés, Susana; Naranjo, José Ramón

    2013-05-31

    Understanding protein interaction networks and their dynamic changes is a major challenge in modern biology. Currently, several experimental and in silico approaches allow the screening of protein interactors in a large-scale manner. Therefore, the bulk of information on protein interactions deposited in databases and peer-reviewed published literature is constantly growing. Multiple databases interfaced from user-friendly web tools recently emerged to facilitate the task of protein interaction data retrieval and data integration. Nevertheless, as we evidence in this report, despite the current efforts towards data integration, the quality of the information on protein interactions retrieved by in silico approaches is frequently incomplete and may even list false interactions. Here we point to some obstacles precluding confident data integration, with special emphasis on protein interactions, which include gene acronym redundancies and protein synonyms. Three human proteins (choline kinase, PPIase and uromodulin) and three different web-based data search engines focused on protein interaction data retrieval (PSICQUIC, DASMI and BIPS) were used to explain the potential occurrence of undesired errors that should be considered by researchers in the field. We demonstrate that, despite the recent initiatives towards data standardization, manual curation of protein interaction networks based on literature searches are still required to remove potential false positives. A three-step workflow consisting of: (i) data retrieval from multiple databases, (ii) peer-reviewed literature searches, and (iii) data curation and integration, is proposed as the best strategy to gather updated information on protein interactions. Finally, this strategy was applied to compile bona fide information on human DREAM protein interactome, which constitutes liable training datasets that can be used to improve computational predictions.

  6. Protein-Protein Interactions: Gene Acronym Redundancies and Current Limitations Precluding Automated Data Integration

    PubMed Central

    Casado-Vela, Juan; Matthiesen, Rune; Sellés, Susana; Naranjo, José Ramón

    2013-01-01

    Understanding protein interaction networks and their dynamic changes is a major challenge in modern biology. Currently, several experimental and in silico approaches allow the screening of protein interactors in a large-scale manner. Therefore, the bulk of information on protein interactions deposited in databases and peer-reviewed published literature is constantly growing. Multiple databases interfaced from user-friendly web tools recently emerged to facilitate the task of protein interaction data retrieval and data integration. Nevertheless, as we evidence in this report, despite the current efforts towards data integration, the quality of the information on protein interactions retrieved by in silico approaches is frequently incomplete and may even list false interactions. Here we point to some obstacles precluding confident data integration, with special emphasis on protein interactions, which include gene acronym redundancies and protein synonyms. Three human proteins (choline kinase, PPIase and uromodulin) and three different web-based data search engines focused on protein interaction data retrieval (PSICQUIC, DASMI and BIPS) were used to explain the potential occurrence of undesired errors that should be considered by researchers in the field. We demonstrate that, despite the recent initiatives towards data standardization, manual curation of protein interaction networks based on literature searches are still required to remove potential false positives. A three-step workflow consisting of: (i) data retrieval from multiple databases, (ii) peer-reviewed literature searches, and (iii) data curation and integration, is proposed as the best strategy to gather updated information on protein interactions. Finally, this strategy was applied to compile bona fide information on human DREAM protein interactome, which constitutes liable training datasets that can be used to improve computational predictions. PMID:28250396

  7. False positive reduction in protein-protein interaction predictions using gene ontology annotations.

    PubMed

    Mahdavi, Mahmoud A; Lin, Yen-Han

    2007-07-23

    Many crucial cellular operations such as metabolism, signalling, and regulations are based on protein-protein interactions. However, the lack of robust protein-protein interaction information is a challenge. One reason for the lack of solid protein-protein interaction information is poor agreement between experimental findings and computational sets that, in turn, comes from huge false positive predictions in computational approaches. Reduction of false positive predictions and enhancing true positive fraction of computationally predicted protein-protein interaction datasets based on highly confident experimental results has not been adequately investigated. Gene Ontology (GO) annotations were used to reduce false positive protein-protein interactions (PPI) pairs resulting from computational predictions. Using experimentally obtained PPI pairs as a training dataset, eight top-ranking keywords were extracted from GO molecular function annotations. The sensitivity of these keywords is 64.21% in the yeast experimental dataset and 80.83% in the worm experimental dataset. The specificities, a measure of recovery power, of these keywords applied to four predicted PPI datasets for each studied organisms, are 48.32% and 46.49% (by average of four datasets) in yeast and worm, respectively. Based on eight top-ranking keywords and co-localization of interacting proteins a set of two knowledge rules were deduced and applied to remove false positive protein pairs. The 'strength', a measure of improvement provided by the rules was defined based on the signal-to-noise ratio and implemented to measure the applicability of knowledge rules applying to the predicted PPI datasets. Depending on the employed PPI-predicting methods, the strength varies between two and ten-fold of randomly removing protein pairs from the datasets. Gene Ontology annotations along with the deduced knowledge rules could be implemented to partially remove false predicted PPI pairs. Removal of false positives

  8. Viable but non-culturable state (VBNC) of Escherichia coli related to EnvZ under the effect of pH, starvation and osmotic stress in sea water.

    PubMed

    Darcan, Cihan; Ozkanca, Reşit; Idil, Onder; Flint, Ken P

    2009-01-01

    When exposed extreme environmental conditions such as sea water, bacteria have been shown different survival strategy for continue their life. One of this strategy known as viable but nonculturable (VBNC) state which is very important for nondifferiation bacteria. VBNC cells cause serious human health problems. Little is known, however, about the genetic mechanisms underlying the VBNC state. Under different environmental conditions, porins are important in the survival strategy of bacteria. EnvZ/OmpR work together as regulators of ompF and ompC gene expression. It is known that the EnvZ system has a role in VBNC state. In this study we tried to find out the viability of EnvZ, OmpC and OmpF mutant E. coli under stress effect of osmolarity, pH and starvation. Bacteria were suspended in filtered-autoclaved sea water microcosms and numbers determined over 25 day incubation periods by plate count (PC), direct viable count (DVC) and count of cells capable of respiration (RCC). As regard to results, alkaline pH affected E. coli more than acidic pH, which led to decline in number. On the contrary glycine betaine addition to sea water protected E. coli porin mutants and also reduced the death rate of bacteria. Under the effect of pH, osmotic stress and starvation stress, wild type E. coli and porin mutants entered a dormant state or became VBNC with the exception of MSZ31 (envZ mutant) E. coli cells which did not enter the VBNC state under the three tested stress conditions. This study is the first report to demonstrate that E. coli could not enter the VBNC state in the lack of EnvZ product under the stress of osmolarity, pH and starvation and the relationship between EnvZ and VBNC state are not affected by pH, osmolarity and starvation.

  9. Functions of BET proteins in erythroid gene expression.

    PubMed

    Stonestrom, Aaron J; Hsu, Sarah C; Jahn, Kristen S; Huang, Peng; Keller, Cheryl A; Giardine, Belinda M; Kadauke, Stephan; Campbell, Amy E; Evans, Perry; Hardison, Ross C; Blobel, Gerd A

    2015-04-30

    Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

  10. pGenN, a Gene Normalization Tool for Plant Genes and Proteins in Scientific Literature

    PubMed Central

    Ding, Ruoyao; Arighi, Cecilia N.; Lee, Jung-Youn; Wu, Cathy H.; Vijay-Shanker, K.

    2015-01-01

    Background Automatically detecting gene/protein names in the literature and connecting them to databases records, also known as gene normalization, provides a means to structure the information buried in free-text literature. Gene normalization is critical for improving the coverage of annotation in the databases, and is an essential component of many text mining systems and database curation pipelines. Methods In this manuscript, we describe a gene normalization system specifically tailored for plant species, called pGenN (pivot-based Gene Normalization). The system consists of three steps: dictionary-based gene mention detection, species assignment, and intra species normalization. We have developed new heuristics to improve each of these phases. Results We evaluated the performance of pGenN on an in-house expertly annotated corpus consisting of 104 plant relevant abstracts. Our system achieved an F-value of 88.9% (Precision 90.9% and Recall 87.2%) on this corpus, outperforming state-of-art systems presented in BioCreative III. We have processed over 440,000 plant-related Medline abstracts using pGenN. The gene normalization results are stored in a local database for direct query from the pGenN web interface (proteininformationresource.org/pgenn/). The annotated literature corpus is also publicly available through the PIR text mining portal (proteininformationresource.org/iprolink/). PMID:26258475

  11. Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

    PubMed

    Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

    2015-12-04

    Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

  12. Eukaryotic expression vectors bearing genes encoding cytotoxic proteins for cancer gene therapy.

    PubMed

    Glinka, Elena M

    2012-09-01

    Cancer gene therapy is a promising direction for the treatment of cancer patients. A primary goal of all cancer therapies is to selectively target and kill tumour cells. Such therapies are administered via different approaches, including both viral and non-viral delivery; however, both methods have advantages and disadvantages. Transcriptional targeting enables genes encoding toxic proteins to be expressed directly in cancer cells. Numerous vectors have been created with the purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Data concerning the function of vectors bearing genes that encode cytotoxic proteins under the control of different promoters, including tissue/tumour specific and constitutive promoters, is summarised here. This review focuses on vectors that bear genes encoding diphtheria toxin, Pseudomonas exotoxin A, caspases, gef, streptolysin, and melittin. Data describing the efficacy of such vectors have been summarised. Notably, there are vectors that killed cancer cell lines originating from the same type of cancer with differential efficiency. Thus, there is differential inhibition of cancer cell growth dependent on the cell line. In this review, the constructs employing genes whose expression induces cell death and the efficiency with which they suppress cancer cell growth will be summarised.

  13. Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

    PubMed Central

    Heath, D G; Cleary, P P

    1989-01-01

    The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins. PMID:2660147

  14. From patenting genes to proteins: the search for utility via function.

    PubMed

    Ilag, Lawrence L; Ilag, Leodegario M; Ilag, Leodevico L

    2002-05-01

    The debate regarding the patenting of genes has extended into the post-genome era. With only approximately 35000 genes deduced from the draft sequence of the human genome, there are fears that a few companies have already gained monopoly on the potential benefits from this knowledge. Nevertheless, it is accepted that proteins determine gene function and function is not readily predicted from gene sequence. Furthermore, genes can encode multiple proteins and a single protein can have multiple functions. Here, we argue that unraveling the intrinsic complexity of proteins and their functions is the key towards determining the utility requirement for patenting protein inventions and consider the possible socioeconomic impact.

  15. Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene.

    PubMed

    Li, Z Y; Song, H Q; Wang, C R; Zhu, X Q

    2016-05-09

    Toxoplasma gondii is a successful opportunistic protozoan distributed worldwide, which can infect all vertebrates, leading to serious infection, blindness, and abortion. Micronemal (MIC) proteins are critically important for T. gondii infection, as they participate in various stages of the Toxoplasma life cycle, including invasion and attachment to host cells. MIC8 secretion relies on the concentration of intracellular calcium, and can mediate the invasion of T. gondii by interacting with soluble MIC3. To investigate genetic diversity of the MIC8 gene, 16 T. gondii strains from different hosts and geographical locations, and two reference isolates (ToxoDB: TGME49_245490 and TGVEG_245490) were examined in this study. The results showed that all the examined MIC8 genes are 2055 bp, with an A+T content ranging from 50.2 to 50.6%. Conversely, lower levels of variation were detected within their nucleotide and amino acid sequences. Phylogenetic analyses indicated that three classical genotypes of T. gondii and the ToxoDB#9 genotype did not group exclusively via Bayesian inference, maximum parsimony, neighbor joining, and/or maximum likelihood assays based on the nucleotide and amino acid sequences of the MIC8 gene. In summary, the T. gondii MIC8 gene is not a suitable marker for population genetic studies of this parasite.

  16. Nuclear genes encoding plastid proteins expressed early in chloroplast development

    SciTech Connect

    Mullet, J.E.

    1991-01-01

    The overall objective of this grant was to characterize events which occur early in chloroplast biogenesis and to isolate nuclear genes encoding plastid proteins which are expressed during this developmental phase. In addition, the possible requirement of plastid transcription for the expression of the nuclear genes such as rbcS and cab was to be tested. The impetus for this research came from studies of chloroplast biogenesis in barley. We found that plastid DNA copy number was relatively high (120 copies/plastid vs 200 at maximal accumulation) in the meristematic region of the leaf base whereas plastid transcription activity was low in this plastid population. Later in chloroplast development transcription activity increased at least 5 fold per plastid or per template indicating that activation of plastid transcription occurred after most of the build up in plastid DNA per plastid. This suggested that activation of plastid DNA synthesis occurred early in chloroplast development and that nuclear genes involved in this activity must be regulated differently from genes such rbcS or cab which are expressed later in development. 3 refs., 7 figs.

  17. A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein immunolocalization.

    PubMed

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2010-03-01

    Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane alpha-helices (80 aa), followed by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane.

  18. [Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap].

    PubMed

    Kopyl, S A; Dorogova, N V; Akhmamet'eva, E M; Omel'ianchuk, L V; Chang, L -S

    2010-03-01

    The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer(DBB) together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.

  19. Targeted genes and interacting proteins of hypoxia inducible factor-1

    PubMed Central

    Liu, Wei; Shen, Shao-Ming; Zhao, Xu-Yun; Chen, Guo-Qiang

    2012-01-01

    Heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) functions as a master regulator of oxygen homeostasis in almost all nucleated mammalian cells. The fundamental process adapted to cellular oxygen alteration largely depends on the refined regulation on its alpha subunit, HIF-1α. Recent studies have unraveled expanding and critical roles of HIF-1α, involving in a multitude of developmental, physiological, and pathophysiological processes. This review will focus on the current knowledge of HIF-1α-targeting genes and its interacting proteins, as well as the concomitant functional relationships between them. PMID:22773957

  20. Mouse Genetic Nomenclature: Standardization of Strain, Gene, and Protein Symbols

    PubMed Central

    Sundberg, John P.; Schofield, Paul N

    2011-01-01

    The use of standard nomenclatures for describing the strains, genes, and proteins of species is vital for the interpretation, archiving, analysis, and recovery of experimental data on the laboratory mouse. At a time when sharing of data and meta- analysis of experimental results is becoming a dominant mode of scientific investigation, failure to respect formal nomenclatures can cause confusion, errors, and in some cases contribute to poor science. Here we present the basic nomenclature rules for laboratory mice and explain how these rules should be applied to complex genetic manipulations and crosses. PMID:20685919

  1. Genes and proteins involved in bacterial magnetic particle formation.

    PubMed

    Matsunaga, Tadashi; Okamura, Yoshiko

    2003-11-01

    Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.

  2. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    networks have been identified, including scale free distribution of the vertex degree, network motifs, and modularity, to name a few. These studies of network organization require the network to be as complete as possible, which given the limitations of experimental techniques is not currently the case. Therefore, experimental procedures for detecting biomolecular interactions should be complemented by computational approaches. The paper by Lees et al provides a review of computational methods, integrating multiple independent sources of data to infer physical and functional protein-protein interaction networks. One of the important aspects of protein interactions that should be accounted for in the prediction of protein interaction networks is that many proteins are composed of distinct domains. Protein domains may mediate protein interactions while proteins and their interaction networks may gain complexity through gene duplication and expansion of existing domain architectures via domain rearrangements. The latter mechanisms have been explored in detail in the paper by Cohen-Gihon et al. Protein-protein interactions are not the only component of the cell's interactome. Regulation of cell activity can be achieved at the level of transcription and involve a transcription factor—DNA binding which typically requires recognition of a specific DNA sequence motif. Chip-Chip and the more recent Chip-Seq technologies allow in vivo identification of DNA binding sites and, together with novel in vitro approaches, provide data necessary for deciphering the corresponding binding motifs. Such information, complemented by structures of protein-DNA complexes and knowledge of the differences in binding sites among homologs, opens the door to constructing predictive binding models. The paper by Persikov and Singh provides an example of such a model in the Cys2His2 zinc finger family. Recent studies have indicated that the presence of such binding motifs is, however, neither necessary

  3. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    PubMed

    Kim, Arthur S; Leaman, Daniel P; Zwick, Michael B

    2014-07-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

  4. Antibody to gp41 MPER Alters Functional Properties of HIV-1 Env without Complete Neutralization

    PubMed Central

    Kim, Arthur S.; Leaman, Daniel P.; Zwick, Michael B.

    2014-01-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design. PMID:25058619

  5. The nucleocapsid protein gene of bovine coronavirus is bicistronic.

    PubMed Central

    Senanayake, S D; Hofmann, M A; Maki, J L; Brian, D A

    1992-01-01

    For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another. Images PMID:1501275

  6. Protein-protein interactions prediction based on iterative clique extension with gene ontology filtering.

    PubMed

    Yang, Lei; Tang, Xianglong

    2014-01-01

    Cliques (maximal complete subnets) in protein-protein interaction (PPI) network are an important resource used to analyze protein complexes and functional modules. Clique-based methods of predicting PPI complement the data defection from biological experiments. However, clique-based predicting methods only depend on the topology of network. The false-positive and false-negative interactions in a network usually interfere with prediction. Therefore, we propose a method combining clique-based method of prediction and gene ontology (GO) annotations to overcome the shortcoming and improve the accuracy of predictions. According to different GO correcting rules, we generate two predicted interaction sets which guarantee the quality and quantity of predicted protein interactions. The proposed method is applied to the PPI network from the Database of Interacting Proteins (DIP) and most of the predicted interactions are verified by another biological database, BioGRID. The predicted protein interactions are appended to the original protein network, which leads to clique extension and shows the significance of biological meaning.

  7. Growing functional modules from a seed protein via integration of protein interaction and gene expression data.

    PubMed

    Maraziotis, Ioannis A; Dimitrakopoulou, Konstantina; Bezerianos, Anastasios

    2007-10-23

    Nowadays modern biology aims at unravelling the strands of complex biological structures such as the protein-protein interaction (PPI) networks. A key concept in the organization of PPI networks is the existence of dense subnetworks (functional modules) in them. In recent approaches clustering algorithms were applied at these networks and the resulting subnetworks were evaluated by estimating the coverage of well-established protein complexes they contained. However, most of these algorithms elaborate on an unweighted graph structure which in turn fails to elevate those interactions that would contribute to the construction of biologically more valid and coherent functional modules. In the current study, we present a method that corroborates the integration of protein interaction and microarray data via the discovery of biologically valid functional modules. Initially the gene expression information is overlaid as weights onto the PPI network and the enriched PPI graph allows us to exploit its topological aspects, while simultaneously highlights enhanced functional association in specific pairs of proteins. Then we present an algorithm that unveils the functional modules of the weighted graph by expanding a kernel protein set, which originates from a given 'seed' protein used as starting-point. The integrated data and the concept of our approach provide reliable functional modules. We give proofs based on yeast data that our method manages to give accurate results in terms both of structural coherency, as well as functional consistency.

  8. The human endogenous retrovirus link between genes and environment in multiple sclerosis and in multifactorial diseases associating neuroinflammation.

    PubMed

    Perron, Hervé; Lang, Alois

    2010-08-01

    Endogenous retroviruses represent about 8% of the human genome and belong to the superfamily of transposable and retrotransposable genetic elements. Altogether, these mobile genetic elements and their numerous inactivated "junk" sequences represent nearly one half of the human DNA. Nonetheless, a significant part of this "non-conventional" genome has retained potential activity. Epigenetic control is notably involved in silencing most of these genetic elements but certain environmental factors such as viruses are known to dysregulate their expression in susceptible cells. More particularly, embryonal cells with limited gene methylation are most susceptible to uncontrolled activation of these mobile genetic elements by, e.g., viral infections. In particular, certain viruses transactivate promoters from endogenous retroviral family type W (HERV-W). HERV-W RNA was first isolated in circulating viral particles (Multiple Sclerosis-associated RetroViral element, MSRV) that have been associated with the evolution and prognosis of multiple sclerosis. HERV-W elements encode a powerful immunopathogenic envelope protein (ENV) that activates a pro-inflammatory and autoimmune cascade through interaction with Toll-like receptor 4 on immune cells. This ENV protein has repeatedly been detected in MS brain lesions and may be involved in other diseases. Epigenetic factors controlling HERV-W ENV protein expression then reveal critical. This review addresses the gene-environment epigenetic interface of such HERV-W elements and its potential involvement in disease.

  9. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  10. Two secretory protein genes in Chironomus tentans have arisen by gene duplication and exhibit different developmental expression patterns.

    PubMed

    Galli, J; Wieslander, L

    1993-05-20

    The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 x 10(4) and 1 x 10(6). Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.

  11. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid. Images PMID:2556372

  12. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed

    Mulbry, W W; Karns, J S

    1989-12-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.

  13. Regulatory elements of Caenorhabditis elegans ribosomal protein genes

    PubMed Central

    2012-01-01

    Background Ribosomal protein genes (RPGs) are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from those of all other species

  14. Parenteral Administration of Capsule Depolymerase EnvD Prevents Lethal Inhalation Anthrax Infection

    PubMed Central

    Negus, David; Vipond, Julia; Hatch, Graham J.; Rayner, Emma L.

    2015-01-01

    Left untreated, inhalation anthrax is usually fatal. Vegetative forms of Bacillus anthracis survive in blood and tissues during infection due to elaboration of a protective poly-γ-d-glutamic acid (PDGA) capsule that permits uncontrolled bacterial growth in vivo, eventually leading to overwhelming bacillosis and death. As a measure to counter threats from multidrug-resistant strains, we are evaluating the prophylactic and therapeutic potential of the PDGA depolymerase EnvD, a stable and potent enzyme which rapidly and selectively removes the capsule from the surface of vegetative cells. Repeated intravenous administration of 10 mg/kg recombinant EnvD (rEnvD) to mice infected with lethal doses of B. anthracis Ames spores by inhalation prevented the emergence of symptoms of anthrax and death; all animals survived the 5-day treatment period, and 70% survived to the end of the 14-day observation period. In contrast to results in sham-treated animals, the lungs and spleen of rEnvD-dosed animals were free of gross pathological changes. We conclude that rEnvD has potential as an agent to prevent the emergence of inhalation anthrax in infected animals and is likely to be effective against drug-resistant forms of the pathogen. PMID:26438506

  15. Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity

    PubMed Central

    Bhattacharyya, Sanchari; Bershtein, Shimon; Yan, Jin; Argun, Tijda; Gilson, Amy I; Trauger, Sunia A; Shakhnovich, Eugene I

    2016-01-01

    Gene dosage toxicity (GDT) is an important factor that determines optimal levels of protein abundances, yet its molecular underpinnings remain unknown. Here, we demonstrate that overexpression of DHFR in E. coli causes a toxic metabolic imbalance triggered by interactions with several functionally related enzymes. Though deleterious in the overexpression regime, surprisingly, these interactions are beneficial at physiological concentrations, implying their functional significance in vivo. Moreover, we found that overexpression of orthologous DHFR proteins had minimal effect on all levels of cellular organization – molecular, systems, and phenotypic, in sharp contrast to E. coli DHFR. Dramatic difference of GDT between ‘E. coli’s self’ and ‘foreign’ proteins suggests the crucial role of evolutionary selection in shaping protein-protein interaction (PPI) networks at the whole proteome level. This study shows how protein overexpression perturbs a dynamic metabolon of weak yet potentially functional PPI, with consequences for the metabolic state of cells and their fitness. DOI: http://dx.doi.org/10.7554/eLife.20309.001 PMID:27938662

  16. Mining topological structures of protein-protein interaction networks for human brain-specific genes.

    PubMed

    Cui, W J; Gong, X J; Yu, H; Zhang, X C

    2015-10-16

    Compared to other placental mammals, humans have unique thinking and cognitive abilities because of their developed cerebral cortex composed of billions of neurons and synaptic connections. As the primary effectors of the mechanisms of life, proteins and their interactions form the basis of cellular and molecular functions in the living body. In this paper, we developed a pipeline for mining topological structures, identifying functional modules, and analyzing their functions from publically available datasets. A human brain-specific protein-protein interaction network with 1482 nodes and 3105 edges was built using a MapReduce based shortest path algorithm. Within this, 7 functional cliques were identified using a network clustering method, 98 hub proteins were obtained by the calculation of betweenness and connectivity, and 5 closest relationship to clique connector proteins were recognized by the combination scores of topological distance and gene ontology similarity. Furthermore, we discovered functional modules interacting with TP53 protein, which involves several fragmented research study conclusions and might be an important clue for further in vivo or in silico experiments to confirm these associations.

  17. Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.

    PubMed

    Wang, Guihua; Wang, Zhenzhen; Zhuang, Pingping; Zhao, Xiaomin; Cheng, Ziqiang

    2017-09-12

    J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J. Copyright © 2017. Published by Elsevier Ltd.

  18. Gene 5 protein-DNA complex: modeling binding interactions.

    PubMed

    Hutchinson, D L; Barnett, B L; Bobst, A M

    1990-08-01

    A helical (not toroidal) complex consisting of eight gene 5 protein dimers per turn is proposed for the extension of DNA from dimer to dimer using known bond length constraints, postulated protein-nucleic acid interactions (determined from NMR and chemical modification studies), other physical properties of the complex, and data from electron micrographs. The binding channel has been dictated by these known parameters and the relative ease of geometrically fitting these constituents. This channel is different from that previously reported by other modelers. The channel lies underneath the long arm "claw-like" extension of the monomer, so that it rests inside the outer surface of the protein complex. An explanation is proposed for the two binding modes, n = 4 (the predominate mode) and n = 3, based on the weak binding interaction of Tyrosine 34. Also, the site of the less mobile nucleic acid base as reported from ESR studies (S.-C. Kao, E.V. Bobst, G.T. Pauly and A.M. Bobst, J. Biom. Struc. Dyn. 3,261 (1985)) is postulated as involving the fourth nucleotide, and this particular base is stacked between Tyrosine 34 and Phenylalanine 73'.

  19. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Treesearch

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  20. Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

    PubMed Central

    Ma, D; Cook, D N; Alberti, M; Pon, N G; Nikaido, H; Hearst, J E

    1993-01-01

    The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed. Images PMID:8407802

  1. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  2. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies.

    PubMed

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC(50) of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors.

  3. Quantifying CD4/CCR5 Usage Efficiency of HIV-1 Env Using the Affinofile System.

    PubMed

    Webb, Nicholas E; Lee, Benhur

    2016-01-01

    Entry of HIV-1 into target cells involves the interaction of the HIV envelope (Env) with both a primary receptor (CD4) and a coreceptor (CXCR4 or CCR5). The relative efficiency with which a particular Env uses these receptors is a major component of cellular tropism in the context of entry and is related to a variety of pathological Env phenotypes (Chikere et al. Virology 435:81-91, 2013). The protocols outlined in this chapter describe the use of the Affinofile system, a 293-based dual-inducible cell line that expresses up to 25 distinct combinations of CD4 and CCR5, as well as the associated Viral Entry Receptor Sensitivity Assay (VERSA) metrics used to summarize the CD4/CCR5-dependent infectivity results. This system allows for high-resolution profiling of CD4 and CCR5 usage efficiency in the context of unique viral phenotypes.

  4. gag, vif, and nef Genes Contribute to the Homologous Viral Interference Induced by a Nonproducer Human Immunodeficiency Virus Type 1 (HIV-1) Variant: Identification of Novel HIV-1-Inhibiting Viral Protein Mutants

    PubMed Central

    D’Aloja, Paola; Olivetta, Eleonora; Bona, Roberta; Nappi, Filomena; Pedacchia, Daniela; Pugliese, Katherina; Ferrari, Giuliana; Verani, Paola; Federico, Maurizio

    1998-01-01

    We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr, pol, or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed

  5. Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes.

    PubMed

    Wang, Jinhua; Smith, Philip J; Krainer, Adrian R; Zhang, Michael Q

    2005-01-01

    Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.

  6. A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression.

    PubMed

    Rodríguez-Mejía, José-Luis; Roldán-Salgado, Abigail; Osuna, Joel; Merino, Enrique; Gaytán, Paul

    2017-01-01

    Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions. © 2016 S. Karger AG, Basel.

  7. Ebolavirus Database: Gene and Protein Information Resource for Ebolaviruses

    PubMed Central

    Sekar, Kanagaraj

    2016-01-01

    Ebola Virus Disease (EVD) is a life-threatening haemorrhagic fever in humans. Even though there are many reports on EVD, the protein precursor functions and virulent factors of ebolaviruses remain poorly understood. Comparative analyses of Ebolavirus genomes will help in the identification of these important features. This prompted us to develop the Ebolavirus Database (EDB) and we have provided links to various tools that will aid researchers to locate important regions in both the genomes and proteomes of Ebolavirus. The genomic analyses of ebolaviruses will provide important clues for locating the essential and core functional genes. The aim of EDB is to act as an integrated resource for ebolaviruses and we strongly believe that the database will be a useful tool for clinicians, microbiologists, health care workers, and bioscience researchers. PMID:27190508

  8. Systematically characterizing and prioritizing chemosensitivity related gene based on Gene Ontology and protein interaction network.

    PubMed

    Chen, Xin; Jiang, Wei; Wang, Qianghu; Huang, Teng; Wang, Peng; Li, Yan; Chen, Xiaowen; Lv, Yingli; Li, Xia

    2012-10-02

    The identification of genes that predict in vitro cellular chemosensitivity of cancer cells is of great importance. Chemosensitivity related genes (CRGs) have been widely utilized to guide clinical and cancer chemotherapy decisions. In addition, CRGs potentially share functional characteristics and network features in protein interaction networks (PPIN). In this study, we proposed a method to identify CRGs based on Gene Ontology (GO) and PPIN. Firstly, we documented 150 pairs of drug-CCRG (curated chemosensitivity related gene) from 492 published papers. Secondly, we characterized CCRGs from the perspective of GO and PPIN. Thirdly, we prioritized CRGs based on CCRGs' GO and network characteristics. Lastly, we evaluated the performance of the proposed method. We found that CCRG enriched GO terms were most often related to chemosensitivity and exhibited higher similarity scores compared to randomly selected genes. Moreover, CCRGs played key roles in maintaining the connectivity and controlling the information flow of PPINs. We then prioritized CRGs using CCRG enriched GO terms and CCRG network characteristics in order to obtain a database of predicted drug-CRGs that included 53 CRGs, 32 of which have been reported to affect susceptibility to drugs. Our proposed method identifies a greater number of drug-CCRGs, and drug-CCRGs are much more significantly enriched in predicted drug-CRGs, compared to a method based on the correlation of gene expression and drug activity. The mean area under ROC curve (AUC) for our method is 65.2%, whereas that for the traditional method is 55.2%. Our method not only identifies CRGs with expression patterns strongly correlated with drug activity, but also identifies CRGs in which expression is weakly correlated with drug activity. This study provides the framework for the identification of signatures that predict in vitro cellular chemosensitivity and offers a valuable database for pharmacogenomics research.

  9. The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

    PubMed Central

    Hamaia, S; Cassé, H; Gazzolo, L; Duc Dodon, M

    1997-01-01

    The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may

  10. Analysis of the fusion protein gene of the porcine rubulavirus LPMV: comparative analysis of paramyxovirus F proteins.

    PubMed

    Berg, M; Bergvall, A C; Svenda, M; Sundqvist, A; Moreno-López, J; Linné, T

    1997-01-01

    Complementary DNA clones representing the fusion (F) protein gene of the porcine rubulavirus LPMV were isolated and sequenced. The F gene was found to be 1,845 nucleotides long containing one long open reading frame capable of encoding a protein of 541 amino acids. The cleavage motif for F0 into F1 and F2 is His-Arg-Lys-Lys-Arg. A sequence comparison and a phylogenetic analysis was performed in order to identify possible functional domains of paramyxovirus fusion proteins and also to classify the porcine rubulavirus. The F gene of LPMV is most closely related to the human mumps virus and simian virus type 5 F genes, and is therefore classified into the rubulavirus genus. A coding region for a small hydrophobic protein was however not found between the F and hemagglutinin-neuraminidase (HN) genes as previously found in both SV5 and mumps.

  11. Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis.

    PubMed Central

    Wang, L F; Doi, R H

    1990-01-01

    The senS gene of Bacillus subtilis, which in high copy number stimulates the expression of several extracellular-protein genes, has been cloned, genetically mapped, and sequenced. The gene codes for a highly charged basic protein containing 65 amino acid residues. The gene is characterized by the presence of a transcription terminator (attenuator) located between the promoter and open reading frame, a strong ribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene. The amino acid sequence of SenS showed partial homology with the N-terminal core binding domain region of bacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins. The gene can be deleted without any effect on growth or sporulation. Images FIG. 7 FIG. 9 PMID:2108127

  12. The human BDNF gene: peripheral gene expression and protein levels as biomarkers for psychiatric disorders

    PubMed Central

    Cattaneo, A; Cattane, N; Begni, V; Pariante, C M; Riva, M A

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) regulates the survival and growth of neurons, and influences synaptic efficiency and plasticity. The human BDNF gene consists of 11 exons, and distinct BDNF transcripts are produced through the use of alternative promoters and splicing events. The majority of the BDNF transcripts can be detected not only in the brain but also in the blood cells, although no study has yet investigated the differential expression of BDNF transcripts at the peripheral level. This review provides a description of the human BDNF gene structure as well as a summary of clinical and preclinical evidence supporting the role of BDNF in the pathogenesis of psychiatric disorders. We will discuss several mechanisms as possibly underlying BDNF modulation, including epigenetic mechanisms. We will also discuss the potential use of peripheral BDNF as a biomarker for psychiatric disorders, focusing on the factors that can influence BDNF gene expression and protein levels. Within this context, we have also characterized, for we believe the first time, the expression of BDNF transcripts in the blood, with the aim to provide novel insights into the molecular mechanisms and signaling that may regulate peripheral BDNF gene expression levels. PMID:27874848

  13. Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1.

    PubMed

    Tokarev, Andrey; Stoneham, Charlotte; Lewinski, Mary K; Mukim, Amey; Deshmukh, Savitha; Vollbrecht, Thomas; Spina, Celsa A; Guatelli, John

    2015-12-16

    HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. Pathogenic viruses encode gene products that enable

  14. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env.

    PubMed

    Heydarchi, Behnaz; Center, Rob J; Bebbington, Jonathan; Cuthbertson, Jack; Gonelli, Christopher; Khoury, Georges; Mackenzie, Charlene; Lichtfuss, Marit; Rawlin, Grant; Muller, Brian; Purcell, Damian

    2017-04-01

    We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.

  15. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env

    PubMed Central

    Center, Rob J.; Bebbington, Jonathan; Cuthbertson, Jack; Khoury, Georges; Lichtfuss, Marit; Rawlin, Grant; Purcell, Damian

    2017-01-01

    ABSTRACT We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection. PMID:27996375

  16. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins

    NASA Astrophysics Data System (ADS)

    Tang, Nicholas C.; Chilkoti, Ashutosh

    2016-04-01

    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  17. (Genetic engineering with a gene encoding a soybean storage protein). Progress report

    SciTech Connect

    Beachy, R.N.

    1985-01-01

    Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the ..cap alpha..'-subunit of ..beta..-conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ACR)

  18. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

    DOEpatents

    Church, George M.; Esvelt, Kevin; Mali, Prashant

    2017-03-07

    Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

  19. Topology association analysis in weighted protein interaction network for gene prioritization

    NASA Astrophysics Data System (ADS)

    Wu, Shunyao; Shao, Fengjing; Zhang, Qi; Ji, Jun; Xu, Shaojie; Sun, Rencheng; Sun, Gengxin; Du, Xiangjun; Sui, Yi

    2016-11-01

    Although lots of algorithms for disease gene prediction have been proposed, the weights of edges are rarely taken into account. In this paper, the strengths of topology associations between disease and essential genes are analyzed in weighted protein interaction network. Empirical analysis demonstrates that compared to other genes, disease genes are weakly connected with essential genes in protein interaction network. Based on this finding, a novel global distance measurement for gene prioritization with weighted protein interaction network is proposed in this paper. Positive and negative flow is allocated to disease and essential genes, respectively. Additionally network propagation model is extended for weighted network. Experimental results on 110 diseases verify the effectiveness and potential of the proposed measurement. Moreover, weak links play more important role than strong links for gene prioritization, which is meaningful to deeply understand protein interaction network.

  20. Functions of milk protein gene 5' flanking regions on human growth hormone gene.

    PubMed

    Ninomiya, T; Hirabayashi, M; Sagara, J; Yuki, A

    1994-03-01

    Fragments containing 5' flanking regions of four bovine milk protein genes--alpha lactalbumin (b alpha LA), alpha S1 casein (b alpha S1CN), beta casein (b beta CN), kappa casein (b kappa CN)--and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5' regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying b alpha LA/, b beta CN/, and mWAP/hGH, and 33% of the lactated b alpha S1CN/hGH rats secreted detectable amounts of hGH (> 0.05 microgram/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of b alpha LA/, b beta CN/, b alpha S1CN/, and mWAP/hGH rats were 1.13-4,360 micrograms/ml, 0.11-10,900 micrograms/ml, 86.8-6,480 micrograms/ml, and 6.87-151 micrograms/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 microgram/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Uncoupling protein 2 gene polymorphisms are associated with obesity

    PubMed Central

    2012-01-01

    Background Uncoupling protein 2 (UCP2) gene polymorphisms have been reported as genetic risk factors for obesity and type 2 diabetes mellitus (T2DM). We examined the association of commonly observed UCP2 G(−866)A (rs659366) and Ala55Val (C > T) (rs660339) single nucleotide polymorphisms (SNPs) with obesity, high fasting plasma glucose, and serum lipids in a Balinese population. Methods A total of 603 participants (278 urban and 325 rural subjects) were recruited from Bali Island, Indonesia. Fasting plasma glucose (FPG), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were measured. Obesity was determined based on WHO classifications for adult Asians. Participants were genotyped for G(−866)A and Ala55Val polymorphisms of the UCP2 gene. Results Obesity prevalence was higher in urban subjects (51%) as compared to rural subjects (23%). The genotype, minor allele (MAF), and heterozygosity frequencies were similar between urban and rural subjects for both SNPs. All genotype frequencies were in Hardy-Weinberg equilibrium. A combined analysis of genotypes and environment revealed that the urban subjects carrying the A/A genotype of the G(−866)A SNP have higher BMI than the rural subjects with the same genotype. Since the two SNPs showed strong linkage disequilibrium (D’ = 0.946, r2 = 0.657), a haplotype analysis was performed. We found that the AT haplotype was associated with high BMI only when the urban environment was taken into account. Conclusions We have demonstrated the importance of environmental settings in studying the influence of the common UCP2 gene polymorphisms in the development of obesity in a Balinese population. PMID:22533685

  2. Learning to Extract Gene-Protein Names from Weakly-Labeled Text

    DTIC Science & Technology

    2008-01-01

    gene -protein entities. We trained a VP -HMM extractor on the training corpus of YAPEX using Minorthird’s default features. The GENIA dataset...Learning to Extract Gene -Protein Names from Weakly-Labeled Text Richard C. Wang, Anthony Tomasic, Robert E. Frederking...Extract Gene -Protein Names from Weakly-Labeled Text 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT

  3. Bacteriophage T5 gene A2 protein alters the outer membrane of Escherichia coli.

    PubMed Central

    Snyder, C E

    1984-01-01

    Evidence for changes in Escherichia coli envelope structure caused by the bacteriophage T5 gene A2 protein was obtained by the use of mutant bacteriophages, envelope fractionation procedures, electrophoretic analysis, and in vitro binding studies with purified gene A2 protein. The results suggested that the T5 gene A2 protein perturbs the host envelope as it functions to promote DNA transfer. Images PMID:6389511

  4. [The study of the uncoulping protein gene as the candidate gene for fatness traits in chicken].

    PubMed

    Zhao, Jian-Guo; Li, Hui; Meng, He; Gu, Zhi-Liang; Wang, Qi-Gui; Wang, Yu-Xiang

    2002-06-01

    The UCPs are integral membrane proteins of the mitochondrial respiration from oxidative phosphorylation, diminishing the resulting production of ATP and instead yielding dissipative heat. The action of those proteins creates a futile cycle that decreases the metabolic efficiency of the organism. Thus UCPs provide new clue to obesity's causes. This study was designed to investigate the effect of UCP gene on chicken fatness traits. The fifth generation population of divergent selection broiler line, Hyline Brown layer and three native breeds (shiqiza, Beijing You, baier) were used in this research. Body weight and body composition traits were measured in broiler lines at 7 weeks of age. Primers for the 3'-untranslator region in UCP were designed from database of chicken genomic sequence. Polymorphisms were detected by PCR-SSCP and DNA sequencing. The results showed that there was significant difference (P < 0.01) in the frequency of genotype among breeds except broiler vs Beijing You and Baier vs Hyline Brown layer in mutation sites detected by the two pairs of primers. The distribution of genotype in Beijing You and broiler had no difference. It deduced that Beijing You belongs to the native breed that has dominant meat type traits and has the same genetic background with broiler. Baier and Hyline Brown Layer have no difference in the genotype, it can be viewed as they have same genetic background. A A/C mutation at base position 1197 was found among individuals in broiler line and the least square analysis showed that BB birds had significant lower (P < 0.01) abdominal fat weight and percentage of abdominal fat than AB or AA birds. From the results we can putatively draw the conclusion that UCP gene is the major gene to affect the fatness traits or it links with the major gene.

  5. Evolutionary hallmarks of the human proteome: chasing the age and coregulation of protein-coding genes.

    PubMed

    Lopes, Katia de Paiva; Campos-Laborie, Francisco José; Vialle, Ricardo Assunção; Ortega, José Miguel; De Las Rivas, Javier

    2016-10-25

    The development of large-scale technologies for quantitative transcriptomics has enabled comprehensive analysis of the gene expression profiles in complete genomes. RNA-Seq allows the measurement of gene expression levels in a manner far more precise and global than previous methods. Studies using this technology are altering our view about the extent and complexity of the eukaryotic transcriptomes. In this respect, multiple efforts have been done to determine and analyse the gene expression patterns of human cell types in different conditions, either in normal or pathological states. However, until recently, little has been reported about the evolutionary marks present in human protein-coding genes, particularly from the combined perspective of gene expression and protein evolution. We present a combined analysis of human protein-coding gene expression profiling and time-scale ancestry mapping, that places the genes in taxonomy clades and reveals eight evolutionary major steps ("hallmarks"), that include clusters of functionally coherent proteins. The human expressed genes are analysed using a RNA-Seq dataset of 116 samples from 32 tissues. The evolutionary analysis of the human proteins is performed combining the information from: (i) a database of orthologous proteins (OMA), (ii) the taxonomy mapping of genes to lineage clades (from NCBI Taxonomy) and (iii) the evolution time-scale mapping provided by TimeTree (Timescale of Life). The human protein-coding genes are also placed in a relational context based in the construction of a robust gene coexpression network, that reveals tighter links between age-related protein-coding genes and finds functionally coherent gene modules. Understanding the relational landscape of the human protein-coding genes is essential for interpreting the functional elements and modules of our active genome. Moreover, decoding the evolutionary history of the human genes can provide very valuable information to reveal or uncover their

  6. Nuclear actin-binding proteins as modulators of gene transcription.

    PubMed

    Gettemans, Jan; Van Impe, Katrien; Delanote, Veerle; Hubert, Thomas; Vandekerckhove, Joël; De Corte, Veerle

    2005-10-01

    Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

  7. Protein and gene model inference based on statistical modeling in k-partite graphs.

    PubMed

    Gerster, Sarah; Qeli, Ermir; Ahrens, Christian H; Bühlmann, Peter

    2010-07-06

    One of the major goals of proteomics is the comprehensive and accurate description of a proteome. Shotgun proteomics, the method of choice for the analysis of complex protein mixtures, requires that experimentally observed peptides are mapped back to the proteins they were derived from. This process is also known as protein inference. We present Markovian Inference of Proteins and Gene Models (MIPGEM), a statistical model based on clearly stated assumptions to address the problem of protein and gene model inference for shotgun proteomics data. In particular, we are dealing with dependencies among peptides and proteins using a Markovian assumption on k-partite graphs. We are also addressing the problems of shared peptides and ambiguous proteins by scoring the encoding gene models. Empirical results on two control datasets with synthetic mixtures of proteins and on complex protein samples of Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana suggest that the results with MIPGEM are competitive with existing tools for protein inference.

  8. Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa▿

    PubMed Central

    Daigle, Denis M.; Cao, Lily; Fraud, Sebastien; Wilke, Mark S.; Pacey, Angela; Klinoski, Rachael; Strynadka, Natalie C.; Dean, Charles R.; Poole, Keith

    2007-01-01

    nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [KD], 158.0 ± 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative “noninteracting” mutant MexR protein, MexRI104F, was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; KD, 853.2 ± 151.1 nM). Consistent with this, MexRI104F repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal α-helix of the

  9. Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

    DOEpatents

    Craft, David L.; Madduri, Krishna M.; Loper, John C.

    2003-01-01

    A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

  10. Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules

    PubMed Central

    Pereira, Lara E.; Clark, Jasmine; Grznarova, Petra; Wen, Xiaoyun; LaCasse, Rachel; Ruml, Tomas; Spearman, Paul

    2014-01-01

    The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4 h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles. PMID:24418544

  11. Functional Gene Group Analysis Indicates No Role for Heterotrimeric G Proteins in Cognitive Ability

    PubMed Central

    Davies, Gail; Liewald, David Cherry McLachlan; Payton, Anthony; Craig, Leone C. A.; Whalley, Lawrence J.; Horan, Mike; Ollier, William; Starr, John M.; Pendleton, Neil; Posthuma, Danielle; Bates, Timothy C.; Deary, Ian J.

    2014-01-01

    Previous functional gene group analyses implicated common single nucleotide polymorphisms (SNPs) in heterotrimeric G protein coding genes as being associated with differences in human intelligence. Here, we sought to replicate this finding using five independent cohorts of older adults including current IQ and childhood IQ, and using both gene- and SNP-based analytic strategies. No significant associations were found between variation in heterotrimeric G protein genes and intelligence in any cohort at either of the two time points. These results indicate that, whereas G protein systems are important in cognition, common genetic variation in these genes is unlikely to be a substantial influence on human intelligence differences. PMID:24626473

  12. Structure-function analysis of the LytM domain of EnvC, an activator of cell wall remodeling at the Escherichia coli division site

    PubMed Central

    Peters, Nick T.; Morlot, Cécile; Yang, Desirée C.; Uehara, Tsuyoshi; Vernet, Thierry; Bernhardt, Thomas G.

    2013-01-01

    Proteins with LytM (Peptidase_M23) domains are broadly distributed in bacteria and have been implicated in a variety of important processes, including cell division and cell-shape determination. Most LytM-like proteins that have been structurally and/or biochemically characterized are metallo-endopeptidases that cleave crosslinks in the peptidoglycan (PG) cell wall matrix. Notable exceptions are the Escherichia coli cell division proteins EnvC and NlpD. These LytM factors are not hydrolases themselves, but instead serve as activators that stimulate PG cleavage by target enzymes called amidases to promote cell separation. Here we report the structure of the LytM domain from EnvC, the first structure of a LytM factor implicated in the regulation of PG hydrolysis. As expected, the fold is highly similar to that of other LytM proteins. However, consistent with its role as a regulator, the active site region is degenerate and lacks a catalytic metal ion. Importantly, genetic analysis indicates that residues in and around this degenerate active site are critical for amidase activation in vivo and in vitro. Thus, in the regulatory LytM factors, the apparent substrate binding pocket conserved in active metallo-endopeptidases has been adapted to control PG hydrolysis by another set of enzymes. PMID:23796240

  13. Dosage Sensitivity of RPL9 and Concerted Evolution of Ribosomal Protein Genes in Plants

    PubMed Central

    Devis, Deborah; Firth, Sue M.; Liang, Zhe; Byrne, Mary E.

    2015-01-01

    The ribosome in higher eukaryotes is a large macromolecular complex composed of four rRNAs and eighty different ribosomal proteins. In plants, each ribosomal protein is encoded by multiple genes. Duplicate genes within a family are often necessary to provide a threshold dose of a ribosomal protein but in some instances appear to have non-redundant functions. Here, we addressed whether divergent members of the RPL9 gene family are dosage sensitive or whether these genes have non-overlapping functions. The RPL9 family in Arabidopsis thaliana comprises two nearly identical members, RPL9B and RPL9C, and a more divergent member, RPL9D. Mutations in RPL9C and RPL9D genes lead to delayed growth early in development, and loss of both genes is embryo lethal, indicating that these are dosage-sensitive and redundant genes. Phylogenetic analysis of RPL9 as well as RPL4, RPL5, RPL27a, RPL36a, and RPS6 family genes in the Brassicaceae indicated that multicopy ribosomal protein genes have been largely retained following whole genome duplication. However, these gene families also show instances of tandem duplication, small scale deletion, and evidence of gene conversion. Furthermore, phylogenetic analysis of RPL9 genes in angiosperm species showed that genes within a species are more closely related to each other than to RPL9 genes in other species, suggesting ribosomal protein genes undergo convergent evolution. Our analysis indicates that ribosomal protein gene retention following whole genome duplication contributes to the number of genes in a family. However, small scale rearrangements influence copy number and likely drive concerted evolution of these dosage-sensitive genes. PMID:26734020

  14. Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription

    PubMed Central

    Deng, Wensheng; Lopez-Camacho, Cesar; Tang, Jen-Yang; Mendoza-Villanueva, Daniel; Maya-Mendoza, Apolinar; Jackson, Dean A.; Shore, Paul

    2012-01-01

    Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease. PMID:22307607

  15. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress.

  16. Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability

    PubMed Central

    Mason, Rosemarie D.; Welles, Hugh C.; Adams, Cameron; Chakrabarti, Bimal K.; Gorman, Jason; Zhou, Tongqing; Nguyen, Richard; O’Dell, Sijy; Lusvarghi, Sabrina; Bewley, Carole A.; Li, Hui; Shaw, George M.; Sheng, Zizhang; Shapiro, Lawrence; Wyatt, Richard; Kwong, Peter D.; Mascola, John R.; Roederer, Mario

    2016-01-01

    The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-27312A. This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy. PMID:27064278

  17. Env sequence determinants in CXCR4-using human immunodeficiency virus type-1 subtype C.

    PubMed

    Lin, Nina H; Becerril, Carlos; Giguel, Francoise; Novitsky, Vladimir; Moyo, Sikhulile; Makhema, Joseph; Essex, Myron; Lockman, Shahin; Kuritzkes, Daniel R; Sagar, Manish

    2012-11-25

    HIV-1 subtype C (HIV-1C) CXCR4-using virus is isolated infrequently and is poorly characterized. Understanding HIV-1C env characteristics has implications for the clinical use of antiretrovirals that target viral entry. A total of 209 env clones derived from 10 samples with mixed CCR5-(R5), CXCR4-using (X4) or dual-tropic HIV-1C were phenotyped for coreceptor usage. Intra-patient X4 and R5 variants generally formed distinct monophyletic phylogenetic clusters. X4 compared to R5 envs had significantly greater amino acid variability and insertions, higher net positive charge, fewer glycosylation sites and increased basic amino acid substitutions in the GPGQ crown. Basic amino acid substitution and/or insertion prior to the crown are highly sensitive characteristics for predicting X4 viruses. Chimeric env functional studies suggest that the V3 loop is necessary but often not sufficient to impart CXCR4 utilization. Our studies provide insights into the unique genotypic characteristics of X4 variants in HIV-1C. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Env sequence determinants in CXCR4-using human immunodeficiency virus type-1 subtype C

    PubMed Central

    Lin, Nina H.; Becerril, Carlos; Giguel, Francoise; Novitsky, Vladimir; Moyo, Sikhulile; Makhema, Joseph; Essex, Myron; Lockman, Shahin; Kuritzkes, Daniel R.; Sagar, Manish

    2012-01-01

    HIV-1 subtype C (HIV-1C) CXCR4-using virus is isolated infrequently and is poorly characterized. Understanding HIV-1C env characteristics has implications for the clinical use of antiretrovirals that target viral entry. A total of 209 env clones derived from 10 samples with mixed CCR5-(R5), CXCR4-using (X4) or dual-tropic HIV-1C were phenotyped for coreceptor usage. Intra-patient X4 and R5 variants generally formed distinct monophyletic phylogenetic clusters. X4 compared to R5 envs had significantly greater amino acid variability and insertions, higher net positive charge, fewer glycosylation sites and increased basic amino acid substitutions in the GPGQ crown. Basic amino acid substitution and/or insertion prior to the crown are highly sensitive characteristics for predicting X4 viruses. Chimeric env functional studies suggest that the V3 loop is necessary but often not sufficient to impart CXCR4 utilization. Our studies provide insights into the unique genotypic characteristics of X4 variants in HIV-1C. PMID:22954962

  19. Bcl-2-related protein family gene expression during oligodendroglial differentiation.

    PubMed

    Itoh, Takayuki; Itoh, Aki; Pleasure, David

    2003-06-01

    Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.

  20. Roles of the Polymerase-Associated Protein Genes in Newcastle Disease Virus Virulence

    PubMed Central

    Yu, Xiao-hui; Cheng, Jin-long; Xue, Jia; Jin, Ji-hui; Song, Yang; Zhao, Jing; Zhang, Guo-zhong

    2017-01-01

    The virulence of Newcastle disease virus varies greatly and is determined by multiple genetic factors. In this study, we systematically evaluated the roles of the polymerase-associated (NP, P and L) protein genes in genotype VII NDV virulence after confirming the envelope-associated (F and HN) proteins contributed greatly to NDV virulence. The results revealed that the polymerase-associated protein genes individually had certain effect on virulence, while transfer of these three genes in combination significantly affected the chimeric virus virulence, especially when the L gene was involved. These results indicated that the L protein was a major contributor to NDV virulence when combined with the homologous NP and P proteins. We also investigated viral RNA synthesis using NDV minigenome systems to assess the interaction between the NP, P, and L proteins, which showed that the activity of the polymerase-associated proteins were directly related to viral RNA transcription and replication. PMID:28220114

  1. Centrin protein and genes in Trichomonas vaginalis and close relatives.

    PubMed

    Brugerolle, G; Bricheux, G; Coffe, G

    2000-01-01

    Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined. By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).

  2. A complete approach for recombinant protein expression training: From gene cloning to assessment of protein functionality*.

    PubMed

    Novo, M Teresa Marques; Soares-Costa, Andrea; de Souza, Antonia Q L; Figueira, Ana Carolina M; Molina, Gustavo C; Palacios, Carlos A; Kull, Claudia R; Monteiro, Izabel F; Baldan-Pineda, Paulo H; Henrique-Silva, Flavio

    2005-01-01

    A practical course was given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering" at the Federal University of São Carlos (UFSCar), São Paulo, Brazil. The goal of the course was to teach current molecular biology tools applied to a real research situation that could be reported by the students themselves. The purpose was to produce a plant recombinant protein and demonstrate a heretofore unreported biological activity. Cystatins, natural inhibitors of cysteine proteases, were proposed for these studies. Initially, the students searched for plant cystatin cDNA sequences in the NCBI databases and selected the Oryzacystatin I gene (ocI) from rice, Oriza sativa, as the target gene for this study. Total RNA was extracted from rice-germinating seeds and primers containing restriction sites for NdeI and EcoRI were designed based on the ocI cDNA sequence and then used to amplify the open reading frame (ORF). RT-PCR amplification provided a band of the expected size for ocI ORF (309 bp). The PCR product was cut with NdeI and EcoRI restriction enzymes and cloned directly in the pET28a expression vector digested with the same enzymes. A pET28-ocI recombinant clone was selected, checked by sequencing, and used to transform Escherichia coli BL21 (DE3) expression strain. After induction of the bacteria with isopropylthiogalactoside and cellular disruption, the His-tagged OCI protein, present mainly in the soluble fraction, was purified by affinity chromatography in a nickel column. The purified protein was successfully used to inhibit fungal growth (Trichoderma reesei). The results were discussed extensively and the students contributed to the writing of this article, of which they are co-authors.

  3. Computer-aided design of modular protein devices: Boolean AND gene activation

    NASA Astrophysics Data System (ADS)

    Salis, H.; Kaznessis, Y. N.

    2006-12-01

    Many potentially useful synthetic gene networks require the expression of an engineered gene if and only if two different DNA-binding proteins exist in sufficient concentration. While some natural and engineered systems activate gene expression according to a logical AND-like behavior, they often utilize allosteric or cooperative protein-protein interactions, rendering their components unsuitable for a toolbox of modular parts for use in multiple applications. Here, we develop a quantitative model to demonstrate that a small system of interacting fusion proteins, called a protein device, can activate an engineered gene according to the Boolean AND behavior while using only modular protein domains and DNA sites. The fusion proteins are created from transactivating, DNA-binding, non-DNA binding, and protein-protein interaction domains along with the corresponding peptide ligands. Using a combined kinetic and thermodynamic model, we identify the characteristics of the molecular components and their rates of constitutive production that maximize the fidelity of AND behavior. These AND protein devices facilitate the creation of complex genetic programs and may be used to create gene therapies, biosensors and other biomedical and biotechnological applications that turn on gene expression only when multiple DNA-binding proteins are simultaneously present.

  4. Avirulence gene mapping in the Hessian fly (Mayetiola destructor) reveals a protein phosphatase 2C effector gene family.

    PubMed

    Zhao, Chaoyang; Shukle, Richard; Navarro-Escalante, Lucio; Chen, Mingshun; Richards, Stephen; Stuart, Jeffrey J

    2016-01-01

    The genetic tractability of the Hessian fly (HF, Mayetiola destructor) provides an opportunity to investigate the mechanisms insects use to induce plant gall formation. Here we demonstrate that capacity using the newly sequenced HF genome by identifying the gene (vH24) that elicits effector-triggered immunity in wheat (Triticum spp.) seedlings carrying HF resistance gene H24. vH24 was mapped within a 230-kb genomic fragment near the telomere of HF chromosome X1. That fragment contains only 21 putative genes. The best candidate vH24 gene in this region encodes a protein containing a secretion signal and a type-2 serine/threonine protein phosphatase (PP2C) domain. This gene has an H24-virulence associated insertion in its promoter that appears to silence transcription of the gene in H24-virulent larvae. Candidate vH24 is a member of a small family of genes that encode secretion signals and PP2C domains. It belongs to the fraction of genes in the HF genome previously predicted to encode effector proteins. Because PP2C proteins are not normally secreted, our results suggest that these are PP2C effectors that HF larvae inject into wheat cells to redirect, or interfere, with wheat signal transduction pathways.

  5. An Introductory Bioinformatics Exercise to Reinforce Gene Structure and Expression and Analyze the Relationship between Gene and Protein Sequences

    ERIC Educational Resources Information Center

    Almeida, Craig A.; Tardiff, Daniel F.; De Luca, Jane P.

    2004-01-01

    We have developed an introductory bioinformatics exercise for sophomore biology and biochemistry students that reinforces the understanding of the structure of a gene and the principles and events involved in its expression. In addition, the activity illustrates the severe effect mutations in a gene sequence can have on the protein product.…

  6. An Introductory Bioinformatics Exercise to Reinforce Gene Structure and Expression and Analyze the Relationship between Gene and Protein Sequences

    ERIC Educational Resources Information Center

    Almeida, Craig A.; Tardiff, Daniel F.; De Luca, Jane P.

    2004-01-01

    We have developed an introductory bioinformatics exercise for sophomore biology and biochemistry students that reinforces the understanding of the structure of a gene and the principles and events involved in its expression. In addition, the activity illustrates the severe effect mutations in a gene sequence can have on the protein product.…

  7. General theory for integrated analysis of growth, gene, and protein expression in biofilms.

    PubMed

    Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S

    2013-01-01

    A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques.

  8. General Theory for Integrated Analysis of Growth, Gene, and Protein Expression in Biofilms

    PubMed Central

    Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S.

    2013-01-01

    A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques. PMID:24376726

  9. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    NASA Astrophysics Data System (ADS)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  10. A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae

    NASA Technical Reports Server (NTRS)

    Hopkins, C. M.; White, F. F.; Heaton, L. A.; Guikema, J. A.; Leach, J. E.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.

  11. A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae

    NASA Technical Reports Server (NTRS)

    Hopkins, C. M.; White, F. F.; Heaton, L. A.; Guikema, J. A.; Leach, J. E.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.

  12. Functional dissection of Odorant binding protein genes in Drosophila melanogaster

    PubMed Central

    Swarup, S; Williams, T I; Anholt, R R H

    2011-01-01

    Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial. PMID:21605338

  13. Polymorphism of prion protein gene in Arctic fox (Vulpes lagopus).

    PubMed

    Wan, Jiayu; Bai, Xue; Liu, Wensen; Xu, Jing; Xu, Ming; Gao, Hongwei

    2009-07-01

    Prion diseases are fatal neurodegenerative disorders of humans and certain other mammals. Prion protein gene (Prnp) is associated with susceptibility and species barrier to prion diseases. No natural and experimental prion diseases have been documented to date in Arctic fox. In the present study, coding region of Prnp from 135 Arctic foxes were cloned and screened for polymorphisms. Our results indicated that the Arctic fox Prnp open reading frame (ORF) contains 771 nucleotides encoding 257 amino acids. Four single nucleotide polymorphisms (SNPs) (G312C, A337G, C541T, and A723G) were identified. SNPs G312C and A723G produced silent mutations, but SNPs A337G and C541T resulted in a M-V change at codon 113 and R-C at codon 181, respectively. The Arctic fox Prnp amino acid sequence was similar to that of the dog (XM 542906). In short, this study provides preliminary information about genotypes of Prnp in Arctic fox.

  14. Tenebrio molitor antifreeze protein gene identification and regulation.

    PubMed

    Qin, Wensheng; Walker, Virginia K

    2006-02-15

    The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

  15. Automatic extraction of gene/protein biological functions from biomedical text.

    PubMed

    Koike, Asako; Niwa, Yoshiki; Takagi, Toshihisa

    2005-04-01

    With the rapid advancement of biomedical science and the development of high-throughput analysis methods, the extraction of various types of information from biomedical text has become critical. Since automatic functional annotations of genes are quite useful for interpreting large amounts of high-throughput data efficiently, the demand for automatic extraction of information related to gene functions from text has been increasing. We have developed a method for automatically extracting the biological process functions of genes/protein/families based on Gene Ontology (GO) from text using a shallow parser and sentence structure analysis techniques. When the gene/protein/family names and their functions are described in ACTOR (doer of action) and OBJECT (receiver of action) relationships, the corresponding GO-IDs are assigned to the genes/proteins/families. The gene/protein/family names are recognized using the gene/protein/family name dictionaries developed by our group. To achieve wide recognition of the gene/protein/family functions, we semi-automatically gather functional terms based on GO using co-occurrence, collocation similarities and rule-based techniques. A preliminary experiment demonstrated that our method has an estimated recall of 54-64% with a precision of 91-94% for actually described functions in abstracts. When applied to the PUBMED, it extracted over 190 000 gene-GO relationships and 150 000 family-GO relationships for major eukaryotes.

  16. Expression of circadian clock genes and proteins in urothelial cancer is related to cancer-associated genes.

    PubMed

    Litlekalsoy, Jorunn; Rostad, Kari; Kalland, Karl-Henning; Hostmark, Jens G; Laerum, Ole Didrik

    2016-07-27

    The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.

  17. Characterizing genes with distinct methylation patterns in the context of protein-protein interaction network: application to human brain tissues.

    PubMed

    Li, Yongsheng; Xu, Juan; Chen, Hong; Zhao, Zheng; Li, Shengli; Bai, Jing; Wu, Aiwei; Jiang, Chunjie; Wang, Yuan; Su, Bin; Li, Xia

    2013-01-01

    DNA methylation is an essential epigenetic mechanism involved in transcriptional control. However, how genes with different methylation patterns are assembled in the p