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1.
The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed. 相似文献
2.
A Hizi L E Henderson T D Copeland R C Sowder H C Krutzsch S Oroszlan 《Journal of virology》1989,63(6):2543-2549
Structural proteins designated p10gag, p21gag, p8gag, p3gag, p27gag, and p14gag from the C3H strain of mouse mammary tumor virus (MMTV) were purified by reversed-phase high-pressure liquid chromatography. The N- and C-terminal amino acid sequences and amino acid composition of each protein were determined and compared with the amino acids encoded by the proviral DNA sequences for the MMTV gag gene. The results show that each of the purified proteins is a proteolytic cleavage product derived from the predicted primary translational product of the gag gene (Pr77gag) and that their order in Pr77gag is p10-pp21-p8-p3-n-p27-p14 (where n represents 17 predicted residues that were not identified among the purified proteins). Purified p10gag lacks the initiator methionine and has a myristoyl group attached in amide linkage to the N-terminal glycine residue predicted by the second codon of the gag gene. The cleavage products are contiguous in the sequence of Pr77gag, and the C-terminal residue of p14gag is encoded by the last codon of the gag gene. By analogy with other retrovirus, p14gag is the viral nucleocapsid protein, p10gag is the matrix protein, and p27gag is the capsid protein of mature MMTV. Proteolytic cleavage sites in MMTV Pr77gag bear a striking resemblance to cleavage sites in the gag precursors of D-type retroviruses, suggesting that these viral proteases have similar specificities. 相似文献
3.
Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins. 总被引:5,自引:16,他引:5
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The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus. 相似文献
4.
Primate retroviruses: intracistronic mapping of type D viral gag gene by use of nonconditional replication mutants.
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Nonconditional replication mutants of squirrel monkey retrovirus (SMRV), an endogenous type D virus of primates, are shown to be defective in post-translational processing of nonglycosylated virus-coded structural proteins. Utilizing such mutants, in combination with sensitive radioimmunological assays, we demonstrate the existence of a 72,000-molecular-weight precursor polyprotein (Pr72gag) encoded by a region of the SMRV genome designated gag. Post-translational cleavage of this precursor polyprotein gives rise to virion structural proteins of 35,000 (p35), 16,000 (p16), 12,000 (p12), and 9,000 (p9) molecular weight. Three of these viral proteins, p35, p16, and p9, are shown to be phosphorylated. Analysis of viral antigen expression in cell lines nonproductively infected with either of two replication-defective SMRV mutants or mink cells productively infected with wild-type SMRV resulted in the detection of several SMRV Pr72gag intermediate cleavage products. Adjacent proteins within such intermediates are identified by use of specific competition immunoassays, and the intracistropic order of individual structural proteins with SMRV Pr72gag was tentatively deduced as NH2-p16-p12-p35-p9-COOH. 相似文献
5.
Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne). 总被引:8,自引:22,他引:8
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L E Henderson R E Benveniste R Sowder T D Copeland A M Schultz S Oroszlan 《Journal of virology》1988,62(8):2587-2595
A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities. 相似文献
6.
Mutational analysis of the murine AIDS-defective viral genome reveals a high reversion rate in vivo and a requirement for an intact Pr60gag protein for efficient induction of disease. 总被引:2,自引:2,他引:0
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Pr60gag appears to be the only protein encoded by the murine AIDS (MAIDS)-defective virus. To study the role of Pr60gag or some other sequences of the viral genome in the pathogenicity of the virus, we have generated mutants of the defective viral genome. These mutant defective viruses, prepared as helper-free stocks, were inoculated into susceptible C57BL/6 mice. Mutant Du5H-A virus, which had a stop codon within gag MA(p15), did not induce target cell proliferation or MAIDS. Mutants Du5H-B and -C encoded truncated Pr60gag proteins containing, respectively, MA(p15)-p12 or MA(p15)-p12 and part of CA(p30). These mutants showed a very limited capacity to induce early cell expansion and were poorly pathogenic. Only recombinant (revertant) viruses were recovered from organs of diseased mice inoculated with these two mutants. Mutant Du5H-D was generated by deleting 1.4 kbp of the 3'-end sequences, outside the gag coding region. The levels of RNA and proteins made by this mutant were low. This mutant also reverting frequently but was nevertheless able to induce MAIDS at a low efficiency without reverting. Our results indicate that the Pr60gag protein is necessary and sufficient to induce MAIDS. These data also suggest that the Pr60gag protein needs to be relatively intact to be fully pathogenic. In addition, our study shows a very high reversion rate of some mutants and emphasizes the need to check for the presence of revertant (recombinant) viruses in diseased organs when working with mutants of the MAIDS-defective virus. 相似文献
7.
Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus 总被引:12,自引:0,他引:12
We have used a recombinant vaccinia virus (VV) which expresses high levels of human immunodeficiency virus-1 (HIV-1) gag proteins to analyze the processing pathway of the gag p55 precursor. HIV-1 gag proteins were isolated from [3H]leucine-labeled VV:gag-infected H9 T lymphocytes by immunoprecipitation with either anti-p24, anti-p17, or anti-p6 antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that processing of the p55 precursor involves three major intermediates (p41a, p41b, and p39). The p41a and p39 proteins contain the p17 and p24 protein segments, and the p41b is comprised of p24 and p15 segments. On two-dimensional gels, each intermediate as well as the mature p24 and p17 proteins migrated as distinct species. [3H]Myristic acid labeling of the HIV-1 gag proteins revealed that in addition to p55 and p17, the p41a and p39 intermediates, but not p41b, are myristylated, confirming that myristylation occurs at the NH2 terminus before cleavage of the p55 precursor protein. We conclude that the myristylated HIV-1 gag p55 precursor is initially cleaved at random either at the p17/p24 junction or at two sites between p24 and p15 proteins, resulting in three intermediates (p41a, p41b, and p39) which are subsequently cleaved to yield mature gag proteins. 相似文献
8.
Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape
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Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein. 相似文献
9.
Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5''-terminal genomic RNA fragment. 总被引:2,自引:3,他引:2
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The retrovirus precursor protein has an arrangement of several characteristic domains with which it achieves selective and efficient packaging of the genome RNA during particle assembly. In this study, we analyzed the composition of the bovine leukemia virus (BLV) gag proteins and examined their RNA-binding properties in gel mobility shift assays, using various genomic RNA probes synthesized in vitro. Results obtained in amino acid sequence and composition analyses indicate that the matrix-associated protein MA(p15) is further processed by the BLV protease (PR) to generate MA(p10), a short peptide of seven amino acid residues, and p4. The gag precursor is now mapped as NH2-MA(p10)-p4-CA(p24)-NC(p12)-COOH. MA(p15) formed a specific complex with the dimer RNA of the U5-5' gag region presumed to contain the BLV packaging signal but not with other RNAs. The NH2-terminal cleavage product, MA(p10), bound all RNA fragments tested, while the COOH-terminal peptides with a sequence common to mammalian type C retroviruses had little affinity for RNA. The nucleocapsid protein NC(p12) bound to RNAs nonspecifically and randomly in the presence or absence of zinc ions. These results suggest a possible interaction of the NH2 terminus of the gag precursor with the 5' terminus of the genomic RNA in an early phase of particle assembly, when the conserved structure between the MA and CA domains might be involved. 相似文献
10.
Amino acid sequence analysis of the proteolytic cleavage products of the bovine immunodeficiency virus Gag precursor polypeptide. 总被引:5,自引:3,他引:2
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G J Tobin R C Sowder nd D Fabris M Y Hu J K Battles C Fenselau L E Henderson M A Gonda 《Journal of virology》1994,68(11):7620-7627
Bovine immunodeficiency virus Gag proteins were purified from virions, and their amino acid sequences and molecular masses were determined. The matrix, capsid, and nucleocapsid (MA, CA, and NC, respectively) and three smaller proteins (p2L, p3, and p2) were found to have molecular masses of 14.6, 24.6, and 7.3 and 2.5, 2.7, and 1.9 kDa, respectively. The order of these six proteins in the Gag precursor, Pr53gag, is NH2-MA-p2L-CA-p3-NC-p2-COOH. In contrast to other retroviral MA proteins, the bovine immunodeficiency virus MA retains its N-terminal methionine and is not modified by fatty acids. In addition, the bovine immunodeficiency virus NC migrates as a 13-kDa protein in denaturing gel electrophoresis; however, its molecular mass was determined to be 7.3 kDa. 相似文献
11.
Mutational analysis of the C-terminal gag cleavage sites in human immunodeficiency virus type 1 总被引:3,自引:0,他引:3
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Coren LV Thomas JA Chertova E Sowder RC Gagliardi TD Gorelick RJ Ott DE 《Journal of virology》2007,81(18):10047-10054
12.
Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products. 总被引:33,自引:28,他引:5
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The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus. 相似文献
13.
Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. 总被引:37,自引:30,他引:7
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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. 相似文献
14.
Bachand F Yao XJ Hrimech M Rougeau N Cohen EA 《The Journal of biological chemistry》1999,274(13):9083-9091
The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag). Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction. Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag). The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions. 相似文献
15.
The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles. 总被引:18,自引:15,他引:3
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The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a virion-associated regulatory protein. Mutagenesis has shown that the virion association of Vpr requires sequences near the C terminus of the HIV-1 Gag polyprotein Pr55gag. To investigate whether Vpr incorporation is mediated by a specific domain of Pr55gag, we examined the ability of chimeric HIV-1/Moloney murine leukemia virus (MLV) Gag polyproteins to direct the incorporation of Vpr. Vpr expressed in trans did not associate with particles formed by the authentic MLV Gag polyprotein or with particles formed by chimeric Gag polyproteins that had the matrix (MA) or capsid (CA) domain of MLV precisely replaced by the corresponding domain of HIV-1HXB2. By contrast, Vpr was efficiently incorporated upon replacement of the C-terminal nucleocapsid (NC) domain of the MLV Gag polyprotein with HIV-1 p15 sequences. Vpr was also efficiently incorporated into particles formed by a MLV Gag polyprotein that had the HIV-1 p6 domain fused to its C terminus. Furthermore, a deletion analysis revealed that a conserved region near the C terminus of the p6 domain is essential for Vpr incorporation, whereas sequences downstream of the conserved region are dispensable. These results show that a virion association motif for Vpr is located within residues 1 to 46 of p6. 相似文献
16.
Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane. 总被引:5,自引:5,他引:0
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To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells. 相似文献
17.
Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. 总被引:44,自引:36,他引:8
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We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag. 相似文献
18.
Differential membrane binding of the human immunodeficiency virus type 1 matrix protein. 总被引:19,自引:14,他引:5
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The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible membrane binding signal to govern its differential subcellular localization and biological functions. We previously identified a membrane binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this membrane binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma membrane binding. To investigate the molecular mechanism for regulation of membrane binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of binding to biological membranes in vitro and in vivo. Liposome binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential membrane binding. 相似文献
19.
20.
R L Shoeman D Young R Pottathil J Victor R R Conroy R M Crowl T Coleman E Heimer C Y Lai K Ganguly 《Analytical biochemistry》1987,161(2):370-379
Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting. 相似文献