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At least 433 records · Page 24Linked to original sources
PubMed2002

Tsg101, an inactive homologue of ubiquitin ligase e2, interacts specifically with human immunodeficiency virus type 2 gag polyprotein and results in increased levels of ubiquitinated gag.

The final stages of budding and release of a retroviral particle from the cell require the late (L) domain of Gag. Recently, ubiquitin and ubiquitin ligases have been implicated in the late stages of retroviral budding. In a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that interact with human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified Tsg101, an inactive homologue of ubiquitin ligase E2. Tsg101 and HIV-2 Gag interact specifically in vitro and in vivo. The interaction requires the L domain PTAPP motif in the p6 domain of HIV-2 Gag and the N-terminal Ubc-conjugation homology domain of Tsg101. Tsg101 is incorporated into HIV-2 virions. Expression of the N-terminal Ubc-conjugation homology domain of Tsg101 inhibits the release of HIV-2 virus particles. Overexpression of Tsg101 results in an increase in the level of ubiquitination of HIV-2 Gag. Our results provide evidence for recruitment of the ubiquitination machinery of the cell during late stages of the viral life cycle, mediated by the viral Gag protein.

Blotting, Western↗
PubMed2002

Molecular organization of Mason-Pfizer monkey virus capsids assembled from Gag polyprotein in Escherichia coli.

We describe the results of a study by electron microscopy and image processing of Gag protein shells-immature capsids--of Mason-Pfizer monkey virus assembled in Escherichia coli from two truncated forms of the Gag precursor: Deltap4Gag, in which the C-terminal p4Gag was deleted, and Pro(-)CA.NC, in which the N-terminal peptides and proline 1 of the CA domain were deleted. Negative staining of capsids revealed small patches of holes forming a trigonal or hexagonal pattern most clearly visible on occasional tubular forms. The center-to-center spacing of holes in the network was 7.1 nm in Deltap4Gag capsids and 7.4 nm in Pro(-)CA.NC capsids. Image processing of Deltap4Gag tubes revealed a hexagonal network of holes formed by six subunits with a single subunit shared between rings. This organization suggests that the six subunits are contributed by three trimers of the truncated Gag precursor. Similar molecular organization was observed in negatively stained Pro(-)CA.NC capsids. Shadowed replicas of freeze-etched capsids produced by either construct confirmed the presence of a hexagonal network of holes with a similar center-to-center spacing. We conclude that the basic building block of the cage-like network is a trimer of the Deltap4Gag or Pro(-)CA.NC domains. In addition, our results point to a key role of structurally constrained CA domain in the trimeric interaction of the Gag polyprotein.

Animals↗
PubMed2003

Vaccinia virus recombinant expressing an 87-kilodalton polyprotein that is sufficient to form astrovirus-like particles.

Human astrovirus is an important cause of acute gastroenteritis. We have generated, for the first time, a vaccinia virus recombinant expressing the astrovirus 87-kDa structural polyprotein. The results demonstrate that this expression results in the formation of virus-like particles in the absence of other astrovirus proteins and genomic RNA. The purified trypsin-activated virus-like particles strongly resemble the complete astrovirus particles.

Base Sequence↗
PubMed2003

Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging.

The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.

Amino Acid Sequence↗
PubMed2005

Inhibition of polyprotein processing and RNA replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions.

Pyrrolidine dithiocarbamate (PDTC) is an antiviral compound that was shown to inhibit the replication of human rhinoviruses (HRVs), poliovirus, and influenza virus. To elucidate the mechanism of PDTC, the effects on the individual steps of the infection cycle of HRV were investigated. PDTC did not interfere with receptor binding or internalization by receptor mediated endocytosis of HRV2 particles into HeLa cells. But we demonstrate that the processing of the viral polyprotein was prevented by PDTC treatment in HeLa cells infected with HRV2. Furthermore, PDTC inhibited the replication of the viral RNA, even when added four hours post infection. As PDTC is described as a metal ion binding agent, we investigated the effect of other metal chelators on the multiplication of HRV2. We show that EDTA, omicron-phenanthroline, and bathocuproine disulfonic acid do not exhibit any antiviral properties. Surprisingly, these substances, coadministered with PDTC, abolished the antiviral effect of PDTC, suggesting that metal ions play a pivotal role in the inhibition of virus multiplication. These results suggest that PDTC inhibits the activity of the viral proteases in a metal ion dependent way.

Antioxidants↗
PubMed1994

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites.

Tetrahymena thermophila micronuclei contain four linker-associated proteins, alpha, beta, gamma, and delta. Synthetic oligonucleotides based on N-terminal protein sequences of beta and gamma were used to clone the micronuclear linker histone (MLH) gene. The MLH gene is single copy and is transcribed into a 2.4-kb message encoding all four linker-associated proteins. The message is translated into a polypeptide (Mic LH) that is processed at the sequence decreases RTK to give proteins whose amino acid sequences differ markedly from each other, from the sequence of macronuclear H1, and from sequences of typical H1s of other organisms. This represents the first example of multiple chromatin proteins derived from a single polyprotein. The delta protein consists largely of two high-mobility-group (HMG) boxes. An evolutionary analysis of HMG boxes indicates that the delta HMG boxes are similar to the HMG boxes of tsHMG, a protein that appears in elongating mouse spermatids when they condense and cease transcription, suggesting that delta could play a similar role in the micronucleus. The micronucleus divides mitotically, while the macronucleus divides amitotically. Surprisingly, macronuclear H1 but not Mic LH contains sequences resembling p34cdc2 kinase phosphorylation sites, while each of the Mic LH-derived proteins contains a typical protein kinase A phosphorylation site in its carboxy terminus.

Amino Acid Sequence↗
PubMed2004

A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon.

Translational initiation of hepatitis C virus (HCV) mRNA occurs by internal entry of ribosomes into an internal ribosomal entry site (IRES) at the 5' nontranslated region. A region encoding the N-terminal part of the HCV polyprotein has been shown to augment the translation of HCV mRNA. Here we show that a cellular protein, NS1-associated protein 1 (NSAP1), augments HCV mRNA translation through a specific interaction with an adenosine-rich protein-coding region within the HCV mRNA. The overexpression of NSAP1 specifically enhanced HCV IRES-dependent translation, and knockdown of NSAP1 by use of a small interfering RNA specifically inhibited the translation of HCV mRNA. An HCV replicon RNA capable of mimicking the HCV proliferation process in host cells was further used to confirm that NSAP1 enhances the translation of HCV mRNA. These results suggest the existence of a novel mechanism of translational enhancement that acts through the interaction of an RNA-binding protein with a protein coding sequence.

Amino Acid Sequence↗
PubMed1990

Fine epitope mapping of the human SS-B/La protein. Identification of a distinct autoepitope homologous to a viral gag polyprotein.

To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library. The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli. A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed. The antigenicities of these recombinant proteins were confirmed with a patient's serum. Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame. Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes. A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined. An additional epitope region (amino acid 179-220) was recognized by some patients' sera. Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein. These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity.

Amino Acid Sequence↗
PubMed2002

Chemical synthesis of NS-1 region of hepatitis C-viral polyprotein fragments: a comparison of PS-BDODMA resin and merrifield resin.

Three peptide fragments selected from the NS-1 region of hepatitis C-viral polyprotein (Leu-Ile-Asn-Thr-Asn-Ala-Ser-Trp-His-Ala-Asn-Arg-Thr-Ala-Leu-Ser Asn-Asp Ser-Lys Leu Asn Thr-Gly Ala NH(2), Leu-lle Asn Thr Asn Ala Ser-Trp-His-Ala-Asn-Arg-Thr Ala NH(2) and Leu-Asn-Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-NH(2)) have been synthesized on PS-BDODMA resin. The synthetic capability of the resin PS-BDODMA resin was compared with Merrifield resin. The peptides were synthesized by the stepwise fluoren-9-yl methoxycarbonyl (Fmoc) solid-phase method. The synthesized peptides were purified by HPLC and the identity of the peptides was established by mass spectrum and amino acid analysis. The synthesis of these peptides illustrates the application of the PS-BDODMA resin for the synthesis of long chain peptides in high yield and homogeneity compared to the Merrifield resin.

Amino Acids↗
PubMed1988

Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs.

The processing of poliovirus precursor polypeptides provides a valuable system in which to study the recognition and interaction of a proteolytic enzyme with its substrates. Processing of the poliovirus polyprotein includes cleavage between 9 of 13 available glutamineglycine (Q-G) pairs by the activity of a virally encoded proteinase, 3C. In this study, we assess the importance of primary, secondary, and tertiary structural determinants in the cleavage at two Q-G pairs in the capsid protein precursor, P1. Employing site-directed mutagenesis of cDNA copies of poliovirus RNA, we have made specific alterations in regions of the P1 capsid precursor and have assayed the effect of these alterations on proteinase cleavage at the two Q-G pairs. We have also introduced additional Q-G pairs into P1 and demonstrated that the proteinase can recognize some of the inserted Q-G pairs as cleavage sites. By correlating the predicted three-dimensional structures and the processing phenotypes of several altered P1 precursors, we are able to rank the importance of determinants required for P1 processing. While a Q-G pair appears to be the primary determinant in proteinase recognition, the tertiary location of a Q-G pair in the precursor either allows or prevents processing at that pair. Our results also suggest that the proper folding of at least two of the three P1 beta-barrel structures is required for efficient proteinase cleavage at Q-G pairs.

Amino Acid Sequence↗
PubMed1994

CTL responses to the gag polyprotein encoded by the murine AIDS defective retrovirus are strain dependent.

Murine AIDS (MAIDS) is induced by a mixture of retroviruses, of which a replication defective virus is the proximal agent of disease. This defective virus harbors a single intact gene that encodes an aberrant gag polyprotein. Certain mouse strains are genetically resistant to MAIDS, with several genes, including H-2Dd, contributing to this resistance. Because MHC class I gene products present intracellular Ags to CTL, recombinant viruses were used to determine whether gag-specific CTLs mediate the genetic linkage between H-2Dd and resistance. Interestingly, while genetically resistant BALB/cByJ and C57BL/KsJ mice (H-2d) generated gag-specific CD8+ CTLs, a similar response was not detected in susceptible BALB.B and C57BL/6J mice (H-2b). However, this CTL response does not appear to be responsible for genetic resistance because: 1) a vigorous CTL response could also be generated by susceptible (C57BL/6 x BALB/cBy) F1 mice and 2) the relevant epitope is H-2Kd restricted.

Animals↗
PubMed1996

Mutations at the capsid-nucleocapsid cleavage site of gag polyprotein of Moloney murine leukemia virus abolish virus infectivity.

Capsid protein CAp30 and nucleocapsid protein NCp10 of Moloney murine leukemia virus (MoMuLV) are the 2 major proteic components of the virion core and are generated by processing of the gag polyprotein precursor, Pr65gag, by the viral protease. In the virion core, several hundred NCp10 molecules are bound to the genomic RNA dimer forming the nucleocapsid structure. In the course of virus assembly, NC protein, as the mature NCp10 and/or as the gag precursor, appears to direct genomic RNA packaging. In vitro, NCp10 has nucleic acid binding and annealing activities and promotes viral RNA dimerization and the annealing of replication primer tRNAPro to the primer binding site (PBS) which is necessary for the initiation of reverse transcription. To investigate whether maturation of NCp10 is required for virus formation, we substituted charged residues for the hydrophobic amino acids at the capsid-nucleocapsid protein cleavage site in order to prevent maturation of NCp10. Here we report that these mutations abolished maturation of capsid protein CAp30 and NCp10 by the viral protease in vitro. When these mutations were introduced into an infectious MoMuLV molecular clone, Pr65gag precursor was synthesized in transfected cells but virion production was strongly diminished and mutant viruses were not infectious. These results suggest that maturation of NCp10 is required for optimal virion release and production of infectious virus.

Binding Sites↗
PubMed1997

Kinetics analysis of consecutive HIV proteolytic cleavages of the Gag-Pol polyprotein.

The ordered, sequential cleavages of the Gag-Pol polyprotein by human immunodeficiency virus (HIV) protease present the virus with severe limitations on viable mutations of the enzyme. An extension of the method of Kuchel et al. (Kuchel, P. W., Nichol, L. W., and Jeffrey, P. D. (1974) J. Theor. Biol. 48, 39-49) for the analysis of consecutive enzyme reactions leads to a simple description of the catalytic efficiency of mutant and wild type HIV protease in the presence or absence of inhibitors. The overall catalytic efficiency of a mutant HIV protease relative to the wild type enzyme is given by the product of the ratios of their respective efficiencies for the 8 obligatory cleavages. Under no conditions is HIV viable when the geometric mean efficiency of a mutant HIV protease is less than 61% of the wild type activity for each cleavage. The lower catalytic efficiencies of the mutant enzymes coupled with the exponential dependence on 1/(1 + [I]/Ki) more than offset the inhibitor resistance acquired by HIV protease. The conclusion of this analysis is that inhibitor-resistant mutant HIV proteases are very unlikely to contribute to viral viability in vivo. The results strongly suggest that future protease inhibitor clinical trials should measure the infectivity of the virions in blood plasma instead of relying on viral RNA levels.

Gene Products, gag↗
PubMed1999

Formation of virus-like particles when the polyprotein gene (segment A) of infectious bursal disease virus is expressed in insect cells.

The baculovirus expression vector system was used to examine the expression of the full-length infectious bursal disease virus (IBDV) segment A cDNA, which encodes the structural proteins in a polyprotein precursor that is autocatalytically cleaved to VPX, VP3, and VP4. No VP2 was observed in lysates of recombinant baculovirus infected cells indicating the lack of processing of VPX to VP2 in this system. Virus-like particles (VLP) were purified from the infected insect cells, and on negative staining electron microscopy, looked very similar to authentic IBDV particles in shape and size, suggesting that processing of VPX to VP2 is not necessary for capsid assembly.

Animals↗
PubMed2005

Detection of antibody to the foot-and-mouth disease virus (FMDV) non-structural polyprotein 3ABC in sheep by ELISA.

The specificity and sensitivity of an ELISA for detecting IgG to the 3ABC non-structural protein of foot-and-mouth disease (FMD) virus was evaluated in FMD naive, aerosol-infected, aerosol plus direct contact infected and field-exposed sheep. All 12 sheep that were experimentally infected without prior vaccination seroconverted in the test, although fewer field sera from FMD-exposed sheep were scored seropositive compared to test results for structural protein antibodies. The 3ABC test specificity was 98 or 100% according to whether sera reacting in the doubtful range were scored as positive or negative. The test was then used to investigate the antibody response of sheep vaccinated against FMD and exposed to the virus by an aerosol challenge 4-14 days later. The response of individual animals varied. Whether immunised with high or low doses of vaccine, the development of 3ABC antibody was most likely in sheep from which live virus was recovered at or beyond 9 days post-challenge. Non-structural responses were also more frequent in animals from which multiple incidences of live FMD virus isolation (perhaps more indicative of true virus replication) were demonstrated.

Animals↗
PubMed2004

Impaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein.

BACKGROUND & AIMS: Multiple molecular mechanisms are likely to contribute to the establishment of persistent infection by hepatitis C virus (HCV). The aim of this work was to study the evasion of cell-mediated antiviral immune responses in transgenic mice with liver-targeted expression of the hepatitis C viral genome. These mice develop steatosis and hepatocellular carcinoma and constitute a murine model of chronic HCV infection. METHODS: Mice of the FL-N/35 lineage were infected with replication-deficient adenoviral vectors encoding beta-galactosidase, and the persistence of infected cells was measured by histochemistry and enzymatic assays. RESULTS: Hepatocytes from the HCV(+) transgenic mice are deficient in eliminating an adenoviral infection, despite an apparently normal T-cell response. The defect in adenoviral clearance was associated with resistance of transgenic hepatocytes to apoptosis induced by Fas/APO1/CD95 death receptor stimulation, a major pathway of cell killing by cytotoxic T lymphocytes. The attenuation of Fas-mediated apoptosis observed in the murine model was associated with a reduced abundance of Bid, a BH3-only member of the Bcl-2 family of apoptosis regulators. CONCLUSIONS: Our results suggest that viral evasion of cell-mediated immune responses leading to apoptotic death of hepatocytes may contribute to viral persistence. Such a mechanism might also contribute to the development of liver cancer in HCV.

Adenoviridae Infections↗