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1.
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
2.
The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. 总被引:41,自引:4,他引:37
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D M D''''Agostino B K Felber J E Harrison G N Pavlakis 《Molecular and cellular biology》1992,12(3):1375-1386
Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs. 相似文献
3.
Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus. 总被引:5,自引:2,他引:3
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Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame. 相似文献
4.
Increased human immunodeficiency virus type 1 (HIV-1) env compartmentalization in the presence of HIV-1-associated dementia
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The human immunodeficiency virus type 1 (HIV-1) surface Env protein has been implicated in the development of HIV-1-associated dementia (HAD). HIV-1 env diversity was analyzed by heteroduplex tracking assay in 27 infected subjects with various neurological statuses. env compartmentalization between the blood and cerebral spinal fluid (CSF) was apparent with all neurological categories. However, in subjects with HAD, significantly more CSF virus was represented by CNS-unique env variants. Variants specialized for replication in the CNS may play a larger role in the development of HAD. Alternatively, HAD may be associated with a more pronounced state of immunosuppression that permits more extensive replication and independent evolution within the CNS compartment. 相似文献
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6.
An infectious chimeric human immunodeficiency virus type 2 (HIV-2) expressing the HIV-1 principal neutralizing determinant. 总被引:1,自引:1,他引:0
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The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND. 相似文献
7.
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
8.
Similarity in env and gag genes between genomic RNAs of human immunodeficiency virus type 1 (HIV-1) from mother and infant is unrelated to time of HIV-1 RNA positivity in the child. 总被引:9,自引:8,他引:1
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G A Mulder-Kampinga A Simonon C L Kuiken J Dekker H J Scherpbier P van de Perre K Boer J Goudsmit 《Journal of virology》1995,69(4):2285-2296
Variation in the env (V3 region) and gag (p17 region) genes of genomic RNA of human immunodeficiency virus type 1 was studied in three mother-child pairs. One infant was human immunodeficiency virus type 1 RNA positive at birth (pair 114), one became positive 6 weeks after birth (pair 127), and one became positive 30 months after birth (pair 564). The first two children were born to seropositive mothers, and the last child was infected by breast-feeding following seroconversion of the mother after delivery. In both V3 and p17gag, intrasample variability was much higher in the maternal samples, including the first seropositive sample of the seroconverted mother, than in the infants' samples. Variability was less in p17gag than in V3, except in the postnatally infected child. In all three cases, infection of the child was established by variants representing a minority of the cell-free virus population in the maternal samples. For the two infants born to seropositive mothers, V3 sequences were more similar to the sequence populations of maternal samples collected during pregnancy than to those of samples collected at delivery or thereafter. However, in pair 114 a V3 variant identical to the child's virus was also detected in the sample collected at delivery. In contrast to the V3 region, p17gag sequences of maternal samples of the first trimester of pregnancy and at delivery had comparable resemblance to the child's sequences in pair 114, while in pair 127, similarity to sequences of the sample collected at delivery was higher than that to sequences of the sample from early in pregnancy. In the last pair, V3 and p17gag sequences from a maternal sample collected 18 months prior to the first RNA-positive sample of the child resembled the infant's sequences as much as the sample collected close to the presumed time of infection. Taken together, the evolutionary characteristics for genomic RNA env and gag genes did not point to a particular time of mother-to-child transmission. 相似文献
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The persistence of human immunodeficiency virus type-1 (HIV-1) has long been attributed to its high mutation rate and the capacity of its resulting heterogeneous virus populations to evade host immune responses and antiviral drugs. However, this view is incomplete because it does not explain how the virus persists in light of the adverse effects mutations in the viral genome and variations in host functions can potentially have on viral functions and growth. Here we show that the resilience of HIV-1 can be credited, at least in part, to a robust response to perturbations that emerges as an intrinsic property of its intracellular development. Specifically, robustness in HIV-1 arises through the coupling of two feedback loops: a Rev-mediated negative feedback and a Tat-mediated positive feedback. By employing a mechanistic kinetic model for its growth we found that HIV-1 buffers the effects of many potentially detrimental variations in essential viral and cellular functions, including the binding of Rev to mRNA; the level of rev mRNA in the pool of fully spliced mRNA; the splicing of mRNA; the Rev-mediated nuclear export of incompletely-spliced mRNAs; and the nuclear import of Tat and Rev. The virus did not, however, perform robustly to perturbations in all functions. Notably, HIV-1 tended to amplify rather than buffer adverse effects of variations in the interaction of Tat with viral mRNA. This result shows how targeting therapeutics against molecular components of the viral positive-feedback loop open new possibilities and potential in the effective treatment of HIV-1. 相似文献
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14.
Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection. 总被引:8,自引:7,他引:1
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The use of recombinant viruses for the expression of a wide array of foreign proteins has become commonplace during the last few years. Recently, we have described the construction and characterization of chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in replication of the RNA genome and expression of HIV-1-P1 fusion proteins (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Here we report on the encapsidation and amplification of the minireplicons to obtain sufficient quantities for biological characterization. To do this, HIV-1-poliovirus minireplicon genomes containing the gag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicated and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidated genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of the HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of the encapsidated chimeric HIV-1-poliovirus genomes was accomplished by coinfecting cells with the encapsidated minireplicons and VV-P1, resulting in stocks of the encapsidated minireplicons. Northern (RNA) blot analysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and type 1 Sabin poliovirus resulted in encapsidation of the chimeric genomes by wild-type poliovirus as measured by immunoprecipitation of the HIV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this system as a potential vaccine vector. 相似文献
15.
Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants. 总被引:6,自引:6,他引:0
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T R Reddy G Kraus O Yamada D J Looney M Suhasini F Wong-Staal 《Journal of virology》1995,69(6):3549-3553
Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected. 相似文献
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17.
Sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type 1 (HIV-1) infection and dendritic-cell-directed HIV-1 transfer
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Both endocytic uptake and viral fusion can lead to human immunodeficiency virus type 1 (HIV-1) transfer to CD4+ lymphocytes, either through directional regurgitation (infectious transfer in trans [I-IT]) or through de novo viral production in dendritic cells (DCs) resulting in a second-phase transfer to CD4+ lymphocytes (infectious second-phase transfer [I-SPT]). We have evaluated in immature monocyte-derived DCs both pathways of transfer with regard to their susceptibilities to being blocked by potential microbicidal compounds, including cyanovirin (CNV); the plant lectins Hippeastrum hybrid agglutinin, Galanthus nivalis agglutinin, Urtica dioica agglutinin, and Cymbidium hybrid agglutinin; and the glycan mannan. I-IT was a relatively inefficient means of viral transfer compared to I-SPT at both high and low levels of the viral inoculum. CNV was able to completely block I-IT at 15 microg/ml. All other compounds except mannan could inhibit I-IT by at least 90% when used at doses of 15 microg/ml. In contrast, efficient inhibition of I-SPT was remarkably harder to achieve, as 50% effective concentration levels for plant lectins and CNV to suppress this mode of HIV-1 transfer increased significantly. Thus, our findings indicate that I-SPT may be more elusive to targeting by antiviral drugs and stress the need for drugs affecting the pronounced inhibition of the infection of DCs by HIV-1. 相似文献
18.
Semen-specific genetic characteristics of human immunodeficiency virus type 1 env 总被引:1,自引:0,他引:1
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Pillai SK Good B Pond SK Wong JK Strain MC Richman DD Smith DM 《Journal of virology》2005,79(3):1734-1742
Human immunodeficiency virus type 1 (HIV-1) in the male genital tract may comprise virus produced locally in addition to virus transported from the circulation. Virus produced in the male genital tract may be genetically distinct, due to tissue-specific cellular characteristics and immunological pressures. HIV-1 env sequences derived from paired blood and semen samples from the Los Alamos HIV Sequence Database were analyzed to ascertain a male genital tract-specific viral signature. Machine learning algorithms could predict seminal tropism based on env sequences with accuracies exceeding 90%, suggesting that a strong genetic signature does exist for virus replicating in the male genital tract. Additionally, semen-derived viral populations exhibited constrained diversity (P < 0.05), decreased levels of positive selection (P < 0.025), decreased CXCR4 coreceptor utilization, and altered glycosylation patterns. Our analysis suggests that the male genital tract represents a distinct selective environment that contributes to the apparent genetic bottlenecks associated with the sexual transmission of HIV-1. 相似文献
19.
Expression of the human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is stringently regulated in infected cells. The majority of the glycoprotein does not reach the cell surface but rather is retained in the endoplasmic reticulum or a cis-Golgi compartment and subsequently degraded. We here report that Env of various HIV-1 isolates is ubiquitinated at the extracellular domain of gp41 and that Env expression could be increased by lactacystin, a specific proteasome inhibitor, suggesting that the ubiquitin/proteasome system is involved in control of expression and degradation. 相似文献
20.
Expression of human immunodeficiency virus type 1 gag modulates ligand-induced downregulation of EGF receptor
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Many enveloped viruses use the ESCRT proteins of the cellular vacuolar protein sorting pathway for efficient egress from the cell. Recruitment of the ESCRT proteins by human immunodeficiency virus type 1 (HIV-1) Gag is required for HIV-1 particle budding and egress. ESCRT proteins normally function at endosomal membranes, where they facilitate the downregulation of mitogen-activated receptors such as EGF receptor (EGFR) through multivesicular body biogenesis. It is not known whether the Gag-mediated recruitment of ESCRT proteins functionally depletes the pool of these molecules that is available for the downregulation of EGFR. Here we show that the expression of HIV-1 Gag decreases the rate of EGFR downregulation, as assessed by decreases in the rates of (125)I-EGF and EGFR degradation. The effect of Gag was dependent on the presence of the TSG101 binding motif (PTAP) within the Gag C-terminal p6 domain. Cells expressing HIV-1 Gag retained more EGFR in late endosomes. This effect occurred when Gag was expressed alone from a heterologous promoter and when Gag expression was driven by the HIV-1 long terminal repeat within pHXB2DeltaBalD25S, a noninfectious lentiviral vector. Gag-expressing cells exhibited higher levels of activated mitogen-activated protein kinase for longer times after EGF addition than did cells that did not express HIV-1 Gag. These results indicate that HIV-1 Gag can impinge upon the functioning of the cellular vacuolar protein sorting pathway and reveal yet another facet of the intricate effects of HIV-1 infection on host cell physiology. 相似文献