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At least 199 records · Page 11Linked to original sources
PubMed1990

Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA.

Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles. To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus. A point mutation in the protease (PR) active site completely prevents Pr76gag processing. The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it. A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA. This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging. These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization.

Amino Acid Sequence↗
PubMed1990

N myristoylation of the spleen necrosis virus matrix protein is required for correct association of the Gag polyprotein with intracellular membranes and for particle formation.

To determine whether myristoylation is required for spleen necrosis virus replication, we constructed a substitution mutation in the gag gene that alters the putative myristate acceptor glycine residue. This single amino acid change was lethal for virus replication, resulted in aberrant proteolytic processing, and interrupted virion assembly and the release of virus from cells. Immunofluorescence analysis indicated that the amount of Gag polyprotein at the cell periphery and in Golgi-associated vesicles is severely reduced in the myristoylation mutant, indicating that correct intracellular targeting is affected by a lack of myristoylation. Coexpression of wild-type Gag polyprotein did not complement and rescue the replication-defective phenotype of the myristoylation mutant. Thus, it appears that the nonmyristoylated polyproteins are incapable of interacting with their myristoylated counterparts to form biologically active particles.

Animals↗
PubMed1991

Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor.

The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay. By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively. We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences. Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I). Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein. This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase.

Amino Acid Sequence↗
PubMed1992

Genetic assay for multimerization of retroviral gag polyproteins.

We have established a genetic assay for the multimerization of retroviral gag polyproteins. This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S. Fields and O. Song, Nature (London) 340:245-246, 1989). In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55gag. The coexpression of these two hybrid proteins in S. cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene. Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins. In contrast, the heterologous GAL4-HIV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression. The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.

Cloning, Molecular↗
PubMed1993

Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays.

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.

Amino Acid Sequence↗
PubMed1993

Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.

Amino Acid Sequence↗
PubMed1995

Active foamy virus proteinase is essential for virus infectivity but not for formation of a Pol polyprotein.

To analyze proteolytic processing of foamy (spuma) retroviruses, two mutations were generated in the presumed active-site triplet Asp-Ser-Gly in the predicted proteinase (PR) region of the human foamy virus (HSRV). The mutations changed either the presumed catalytic aspartic acid residue to a catalytically incompetent alanine or the adjacent serine to a threonine found in most cellular and retroviral proteases at this position. Both mutations were cloned into the full-length infectious HSRV DNA clone. Wild-type and S/T mutant genomes directed the synthesis of particles with similar infectious titers, while the HSRV D/A PR mutant was noninfectious. Immunoblot analysis of transfected cells revealed identical patterns for the wild-type and for the S/T PR mutant. HSRV D/A mutant-transfected cells expressed only a single Gag polyprotein of 78 kDa instead of the 78-kDa-74-kDa doublet found in HSRV-infected or wild-type-transfected cells. Analysis with pol-specific antisera yielded a protein of approximately 120 kDa reactive with antisera against pol- but not gag-specific domains. No Gag-Pol polyprotein was detected in this study. Electron microscopy analysis of transfected cells showed heterogeneous particle morphology in the case of the D/A mutant, with particles of normal appearance and particles of aberrant size and shape. These results indicate that foamy viruses have an aspartic PR that is essential for infectivity but not for formation of the 120-kDa Pol polyprotein.

Amino Acid Sequence↗
PubMed1995

Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication.

The determinants of cleavage site specificity of the yellow fever virus (YF) NS3 proteinase for its 2B/3 cleavage site have been studied by using site-directed mutagenesis. Mutations at residues within the GARR decreases S sequence were tested for effects on cis cleavage of an NS2B-3(181) polyprotein during cell-free translation. At the P1 position, only the conservative substitution R-->K exhibited significant levels of cleavage. Conservative and nonconservative substitutions were tolerated at the P1' and P2 positions, resulting in intermediate levels of cleavage. Substitutions at the P3 and P4 positions had no effects on cleavage efficiency in the cell-free assay. Processing at other dibasic sites was studied by using transient expression of a sig2A-5(356) polyprotein. Cleavage at the 2B/3 site was not required for processing at downstream sites. However, increased accumulation of high-molecular-weight viral polyproteins was generally observed for mutations which reduced cleavage efficiency at the 2B/3 site. Several mutations were also tested for their effects on viral replication. Virus was not recovered from substitutions which blocked or substantially reduced cleavage in the cell-free assay, suggesting that efficient cleavage at the 2B/3 site is required for flavivirus replication.

Amino Acid Sequence↗
PubMed1997

Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation.

Proteolytic processing of the Rous sarcoma virus (RSV) Gag precursor was altered in vivo through the introduction of amino acid substitutions into either the polyprotein cleavage junctions or the PR coding sequence. Single amino acid substitutions (V(P2)S and P(P4)G), which are predicted from in vitro peptide substrate cleavage data to decrease the rate of release of PR from the Gag polyprotein, were placed in the NC portion of the NC-PR junction. These substitutions do not affect the efficiency of release of virus-like particles from COS cells even though recovered particles contain significant amounts of uncleaved Pr76gag in addition to mature viral proteins. Single amino acid substitutions (A(P3)F and S(P1)Y), which increase the rate of PR release from Gag, also do not affect budding of virus-like particles from cells. Substitution of the inefficiently cleaved MA-p2 junction sequence in Gag by eight amino acids from the rapidly cleaved NC-PR sequence resulted in a significant increase in cleavage at the new MA-p2 junction, but again without an effect on budding. However, decreased budding was observed when the A(P3)F or S(P1)Y substitution was included in the NC-PR junction sequence between the MA and p2 proteins. A budding defect was also caused by substitution into Gag of a PR subunit containing three amino acid substitutions (R105P, G106V, and S107N) in the substrate binding pocket that increase the catalytic activity of PR. The defect appears to be the result of premature proteolytic processing that could be rescued by inactivating PR through substitution of a serine for the catalytic aspartic acid residue. This budding defect was also rescued by single amino acid substitutions in the NC-PR cleavage site which decrease the rate of release of PR from Gag. A similar budding defect was caused by replacing the Gag PR with two PR subunits covalently linked by four glycine residues. In contrast to the defect caused by the triply substituted PR, the budding defect observed with the linked PR dimer could not be rescued by NC-PR cleavage site mutations, suggesting that PR dimerization is a limiting step in the maturation process. Overall, these results are consistent with a model in which viral protein maturation occurs after PR subunits are released from the Gag polyprotein.

Animals↗
PubMed1997

Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits virus spread.

The antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly. Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro. A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold. A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins. The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold. An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.

3T3 Cells↗
PubMed1998

Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.

Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448-1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDpro cleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins > 10 kDa in size through fusion with the polyprotein needs to be reevaluated.

Amino Acid Sequence↗
PubMed1998

Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate.

Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitro trans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus ppla and pplab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.

Amino Acid Sequence↗
PubMed1998

A proline-rich motif (PPPY) in the Gag polyprotein of Mason-Pfizer monkey virus plays a maturation-independent role in virion release.

Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the gag gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.

Animals↗
PubMed1999

Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsid-dimer interface and the basic region of matrix protein.

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Many gag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in trans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.

Animals↗
PubMed1999

Actin associates with the nucleocapsid domain of the human immunodeficiency virus Gag polyprotein.

Recently, it was shown that actin molecules are present in human immunodeficiency virus type 1 (HIV-1) particles. We have examined the basis for incorporation and the location of actin molecules within HIV-1 and murine retrovirus particles. Our results show that the retroviral Gag polyprotein is sufficient for actin uptake. Immunolabeling studies demonstrate that actin molecules localize to a specific radial position within the immature particle, clearly displaced from the matrix domain underneath the viral membrane but in proximity to the nucleocapsid (NC) domain of the Gag polyprotein. When virus or subviral Gag particles were disrupted with nonionic detergent, actin molecules remained associated with the disrupted particles. Actin molecules remained in a stable complex with the NC cleavage product (or an NC-RNA complex) after treatment of the disrupted HIV-1 particles with recombinant HIV-1 protease. In contrast, matrix and capsid molecules were released. The same result was obtained when mature HIV-1 particles were disrupted with detergent. Taken together, these results indicate that actin molecules are associated with the NC domain of the viral polyprotein.

Actins↗
PubMed1999

The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site.

The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.

Animals↗
PubMed1986

[2 nonstructural glycosylated polyproteins coded by the gag gene of retrovirus type D].

Two nonstructural glycosylated gag-encoded polyproteins were identified in D type retrovirus-infected cells of human transplantable laryngeal carcinoma by a "pulse-chase" modification of the immunoprecipitation method. The molecular weights of these polyproteins were 78 K (gPr 78gag) and 90 K (gPr 90gag), respectively. Glucosamine and galactose constituted their carbohydrate component. Initially, gPr 90gag is synthesized, to be followed by gPr 78gag, perhaps due to the split of gPr 90gag. The functional role of these glycoproteins remains to be established. It is suggested that the said and similar glycosylated polyproteins of other retroviruses play a role in stimulating proliferation of retrovirus-infected cells.

Chemical Precipitation↗
PubMedDate not supplied

[Nonstructural glycosylated polyproteins coded for by the gag gene of retrovirus type D expressed on the plasma membrane].

Studies of virus-specific proteins of type D retroviruses by radioimmunoprecipitation (RIP) using various radioactive precursors demonstrated in HEp-2 cells producing these retroviruses the presence of two glycosylated gag-coded polyproteins with molecular weights of 78 K and 90 K. The same polyproteins were found by lactoperoxidase iodination and RIP on the plasma membrane of the cells. Comparative studies of these glycoproteins by the limited proteolysis method showed the sites for trypsin action in them to be located in similar places of protein bases which indicated their structural relationship. It is suggested that glycosylated gag-coded polyproteins of retroviruses (including type D retroviruses) play the role of receptors for certain mitogens the seizure of which stimulates cell proliferation.

Electrophoresis, Polyacrylamide Gel↗