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1.
J Biol Chem ; 276(8): 5985-91, 2001 Feb 23.
Article in English | MEDLINE | ID: mdl-11054427

ABSTRACT

HIV (human immunodeficiency virus)-1 Env is displayed on the surface of infected cells and subsequently incorporated into virions, which is necessary for the initiation of a viral infection by recognition of the CD4 and the chemokine receptors (such as CCR5 or CXCR4) on the surface of new target cells. As a type 1 integral membrane glycoprotein, Env is cotranslationally translocated into the endoplasmic reticulum. In this report, we characterized the synthesis of Env, which did not occur at a constant rate but by translational/translocational pausing that has not previously been shown with a viral encoded glycoprotein. Overall translation was not impeded by the presence of the reducing agent dithiothreitol in vivo, although this did influence the cleavage of the precursor gp160 into its mature form, gp120. Env interacts transiently with resident components of the endoplasmic reticulum such as calnexin, which had maximal association at a 10-min post-translation. Addition of the glucosidase inhibitor, castanospermine, failed to significantly influence the association of Env with calnexin, consistent with the notion that calnexin recognizes components other than alpha-terminal glucose. Moreover, castanospermine treatment failed to affect the infectivity of virions. Taken together, this report demonstrates the existence of translational/translocational pausing for a viral glycoprotein and suggests that trimming of glucose from HIV-1 Env is not essential for the initiation of virus infection.


Subject(s)
Glucose/pharmacology , HIV Envelope Protein gp160/biosynthesis , HIV-1/pathogenicity , Protein Processing, Post-Translational/drug effects , Protein Transport/drug effects , T-Lymphocytes/virology , Adenosine Triphosphate/metabolism , Calcium-Binding Proteins/metabolism , Calnexin , Disulfides/metabolism , Dithiothreitol/pharmacology , Endoplasmic Reticulum/metabolism , Glycosylation , Indolizines/pharmacology , Protein Binding , Protein Biosynthesis , Virion/pathogenicity
2.
J Virol ; 74(19): 8938-45, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10982337

ABSTRACT

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA(3)(Lys) were additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced in vif mutant virions. Moreover, purified vif mutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.


Subject(s)
Gene Products, vif/genetics , HIV-1/physiology , RNA, Viral/genetics , HIV Reverse Transcriptase/genetics , Humans , Transcription, Genetic , Virus Replication , vif Gene Products, Human Immunodeficiency Virus
3.
J Virol ; 73(11): 9145-52, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10516021

ABSTRACT

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are an important parameter of host defenses that limit viral replication after infection. Induction of effective CTL against conserved viral proteins such as Gag may be essential to the development of a safe and effective HIV type 1 (HIV-1) vaccine. DNA vaccination represents a novel strategy for inducing potent CD8(+) CTL responses in vivo. However, expression of HIV-1 structural proteins by DNA vectors has been hampered by a stringent requirement for coexpression with other viral components, such as Rev and RRE. Furthermore, even with Rev and RRE present, the level of expression of HIV-1 Gag, Pol, or Env is very low in murine cells. These problems have limited our ability to address the key issue of how to generate effective CTL responses to Gag in a mouse model. To overcome this problem, we compared several novel DNA expression vectors for HIV-1 Gag protein expression in primate and mouse cells and for generating immune responses in mice after DNA vaccination. A DNA vector containing wild type HIV-1 gag coding sequences did not induce detectable Gag expression in any of the cells tested. Attempts to increase nuclear export of Gag expression RNA by adding the constitutive transport element yielded only a moderate increase in Gag expression in monkey-derived COS cells and an even lower increase in Gag expression in HeLa cells or several mouse cell lines. In contrast, silent-site mutations in the HIV-1 gag coding sequences significantly increased Gag expression levels in all cells tested. Furthermore, this construct induced both Gag-specific antibody and CTL responses in mice after DNA vaccination. Using this construct, we achieved stable expression of HIV-1 Gag in the mouse cell line p815, which can now be used as a target cell for measuring HIV-1 Gag-specific CTL responses in immunized mice. The DNA vectors described in this study should make it possible to systematically evaluate the approaches for maximizing the induction of CTL responses against HIV-1 Gag in mouse and other animal systems.


Subject(s)
AIDS Vaccines/immunology , Gene Products, gag/genetics , Gene Products, gag/immunology , HIV-1/genetics , HIV-1/immunology , Vaccines, DNA/immunology , Animals , Cell Line , Evaluation Studies as Topic , Gene Products, gag/biosynthesis , Genes, gag , Genetic Vectors , HIV Antibodies/blood , HIV Infections/prevention & control , HIV Infections/virology , Humans , Immunoblotting , Mice , Mice, Inbred BALB C , Protein Precursors/biosynthesis , Protein Precursors/genetics , Protein Precursors/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccination
4.
J Virol ; 73(6): 4696-704, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10233929

ABSTRACT

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6(Gag). This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412-3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6(Gag) resulted in a cell type-dependent replication of the mutant virus in CD4(+) T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6(Gag) mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an approximately 16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160(Gag-Pol) or in the interaction between p55(Gag) and p160(Gag-Pol) precursors. Electron microscopic analysis of the p6(Gag) mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6(Gag) is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.


Subject(s)
Gene Products, gag/physiology , Gene Products, pol/physiology , HIV-1/physiology , Virion/physiology , Virus Replication , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Fusion Proteins, gag-pol/metabolism , Gene Products, gag/metabolism , Microscopy, Electron , Molecular Sequence Data , Proline , Protein Precursors/metabolism , Structure-Activity Relationship , Virus Assembly
5.
J Virol ; 73(2): 1460-7, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9882352

ABSTRACT

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.


Subject(s)
Gene Products, vif/isolation & purification , HIV-1 , Animals , COS Cells , Centrifugation, Density Gradient , Humans , Virion , vif Gene Products, Human Immunodeficiency Virus
6.
J Immunol ; 161(11): 6406-12, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9834132

ABSTRACT

The factors controlling the dynamics of HIV-1 transmission from mother to infant are not clearly known. Previous studies have suggested the existence of maternal and placental protective mechanisms that inhibit viral replication in utero. Preliminary studies from our laboratory revealed that supernatant from placental stromal cells protected HIV-1-infected PBMC from virus-induced apoptosis and suppressed virus production. We have attempted to characterize the antiviral activity of this placental factor (PF) and delineate the stages of HIV-1 replication affected. This activity was not due to the presence of any known cytokine reported to have anti-HIV effect. Direct exposure to PF had no suppressive effect on the infectivity of cell-free HIV-1, and envelope-mediated membrane fusion appeared to be unaffected. Western blot analysis of HIV-1 from infected PBMC treated with PF revealed that expression of all viral proteins was reduced proportionately, both intracellularly and in released virions. However, exposure of HIV-1-infected cells to PF resulted in production of virions with 10-100-fold-reduced infectivity. PF-treated virions contained two- to threefold reduced ratios of cyclophilin A:Gag protein as compared with untreated virus. Reduced cyclophilin A content resulting in decreased binding of cyclophilin A to Gag could account, in part, for the observed reduction in infectivity. Our results suggest that placental cells produce an antiviral factor that protects the fetus during gestation and may have therapeutic potential.


Subject(s)
Anti-HIV Agents/pharmacology , Antiviral Agents/biosynthesis , Antiviral Agents/physiology , HIV-1/growth & development , Placenta/metabolism , Pregnancy Proteins/biosynthesis , Pregnancy Proteins/physiology , Cell Fusion/immunology , Cells, Cultured , Chemical Phenomena , Chemistry, Physical , HeLa Cells , Humans , Placenta/cytology , Stromal Cells/cytology , Stromal Cells/metabolism , Viral Proteins/analysis , Virion/chemistry , Virion/pathogenicity , Virus Replication/immunology
7.
J Virol ; 72(4): 3412-7, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9525672

ABSTRACT

One of the crucial steps in the assembly of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses is the incorporation and retention of all the key viral enzymes in released virions. The viral enzymes protease, reverse transcriptase, and integrase of HIV-1 are initially synthesized as Gag-Pol fusion polyproteins. It has been shown that the incorporation of Gag-Pol polyproteins during virus assembly requires the Gag domains that are shared by the Gag and Gag-Pol precursors. We now report that truncation of the C-terminal p6 domain of HIV-1 Gag, which is present in the Gag precursor but not in the Gag-Pol precursor, drastically reduced the amount of Pol proteins in the mutant virions. Mutations in the lentivirus conserved motif P(T/S)APP in p6 also drastically reduced the amount of Pol proteins in mutant virions. The steady-state levels of Gag-Pol precursors and cleaved Pol proteins in the transfected cells were not affected by mutations in p6. The incorporation of unprocessed Gag-Pol precursors into p6 mutant virions was detected when the viral protease was mutated, suggesting that the interactions among mutant Gag molecules and Gag-Pol precursors were not significantly affected. These results suggest that the p6 domain of HIV-1 Gag may play an important role in recruiting or retaining cleaved Pol proteins during virus assembly.


Subject(s)
Gene Products, gag/genetics , Gene Products, gag/physiology , Gene Products, pol/metabolism , HIV-1/physiology , Mutation , Virus Assembly , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , COS Cells , Fusion Proteins, gag-pol/metabolism , HIV Protease/genetics , HIV Protease/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , HIV-1/metabolism , Humans , Intracellular Fluid/metabolism , Molecular Sequence Data , Mutagenesis , Point Mutation , Protein Processing, Post-Translational , Virion , gag Gene Products, Human Immunodeficiency Virus
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