Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study.

We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was far much higher than those of an APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. We finally monitored the incorporation of APOBEC3G into virions produced in A3G-C1. APOBEC3G was easily detected in progeny viral particles upon transfection of vif-minus proviral clone but not of wild type. These results indicated that our new A3G-C1 cell line is eminently useful for various studies on the interaction of human APOBEC3G and HIV-1 Vif.

Unique characteristics of HIV-1 Vif expression.

We examined the steady-state expression in cells of four accessory proteins of human immunodeficiency virus type 1 (HIV-1). For this purpose, a series of single gene expression vectors for these viral proteins were constructed and were monitored for their production by transfection. Among them, the expression level of Vif was found to be lowest in both the absence and presence of APOBEC3G. In addition, we noticed the presence of its truncated form, which was not observed for the other accessory proteins. When a subgenomic vector was used for transfection, authentic and several small forms of Vif were produced. By mutational analysis, these forms were demonstrated to be mutant Vif proteins translated from M8, M16 and M29. When a full-length molecular clone was used, the smaller versions of Vif were hardly observed. Functional analysis of these mutant Vif proteins showed that they are incapable of modulating viral infectivity. The results described above, i.e. the low steady-state expression and the presence of truncated forms, represent the unique characteristics of HIV-1 Vif.

Human immunodeficiency virus type 1 Vif supports efficient primate lentivirus replication in rhesus monkey cells.

Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) Vif share limited homology and display species-specific activity, leading to speculation that Vif sequences could determine the block in HIV-1 replication in rhesus monkeys. To address this issue, we engineered a novel SIV recombinant in which HIV-1 vif replaced SIV vif in a SIVmac239 background. Insertion of HIV-1 vif into the SIV vif locus did not produce a replication-competent virus. Therefore, we inserted HIV-1 vif sequences into the SIV nef locus, which produced a recombinant that, in the absence of SIV vif sequences, replicated similarly to wild-type SIVmac239 in rhesus monkey PBMC. From these studies we conclude that the HIV-1 replication block in rhesus monkeys is almost certainly not Vif determined. These studies also suggest that SHIV/NVif or derivative sequences could be utilized for structure/function studies of HIV-1 Vif in experimentally infected rhesus monkeys.

[The function of accessory genes of HIV-1].

Human immunodeficiency virus type 1 (HIV-1) has 4 auxiliary genes, vpr, vpu, nef, and vif, which are dispensable for viral replication in vitro. However, many studies with animal model revealed that these genes play important roles on the viral replication and the development of AIDS in vivo through many complicated mechanisms. Although several key factors involved in the function have been identified, further studies are required for the complete understandings of the action mechanisms. The elucidation of the function of the auxiliary genes on molecular bases leads to the discovery of new therapeutic strategies against HIV and the understanding of basic cellular mechanisms. In this review, we summarize new observations mainly about the interactions between auxiliary genes and host cell functions.

Aspectos biomoleculares de la infección por el virus de la inmunodeficiencia humana / Biomolecular aspects of the human immunodeficiency virus infection

El presente artículo de revisión se centra en las consideraciones fundamentales sobre la biología molecular del virus de la inmunodeficiencia humana, el principal de los retrovirus que infectan a los hombres, destacando sus aspectos constitutivos así como genéticos y las alteraciones con la célula huésped. Se presenta además los aspectos morfofuncionales básicos de los linfocitos T CD4 y las interacciones que a nivel de membrana se producen para favorecer la infección por el virus y que explican su fisiopatología. Finalmente, se destacan los eventos intracelulares que conducen a la replicación y ensamblaje viral que producen la muerte celular y explican la inmunosupresión del huésped

Cell-free HIV-1Zr6 vif mutants are defective in binding to peripheral blood mononuclear cells and in internalization.

The vif gene of the human immunodeficiency virus (HIV-1) is required for productive virus infection of primary blood mononuclear cells (PBMCs) and macrophages in vitro. Replication of HIV-1 vif- mutants in T-lymphoid cell lines varies and is dependent on the cell line used for virus production. To further understand the role of Vif in HIV-1 infection, we constructed to vif deletion mutants from a molecular clone derived from an African patient (HIV-1Zr6). Cell-free Zr6 vif- virus pools made from transfected rhabdomyosarcoma (RD) cells do not replicate when added to cultures of stimulated PBMCs. However, vif mutants were able to spread from transfected RD cells to PBMCs if cell-to-cell contact was permitted. By Western blot analysis, viral structural proteins expressed after transfection of RD cells by wild-type or vif mutant proviruses were indistinguishable. However, binding of vif mutants to PBMCs or to purified CD4 and virus internalization were significantly reduced when compared with wild-type virus. The defects in cell-free infection, CD4 binding, and internalization were rescued by transcomplementation using a vif expression plasmid. Our results suggest a novel level at which the HIV-1 vif gene product acts to enhance cell-free infection and indicate that vif plays an important role in promoting HIV-1 binding and internalization. Combined with the previous reports of vif's effect at other steps in infection, this suggests that vif is a pleuripotent gene product that affects multiple stages of the infective process.

The requirement for Vif of SIVmac is cell-type dependent.

The vif gene (viral infectivity factor) of the human and simian immunodeficiency viruses (HIV and SIV) is present in almost all members of the lentivirus group of retroviruses. This gene is highly conserved among different HIV and SIV isolates and is therefore presumed to play an important role in pathogenesis. To analyse the role of Vif in SIV, three SIVmac mutants have been constructed by introducing site-specific mutations or deletions into vif of the pathogenic molecular clone SIVmac239. The effect of Vif on viral replication in T cells was examined by transfecting equal amounts of either vif-positive or vif-negative viral DNA into SupT1, CEM-SS and H9 cells. Reverse transcriptase assay of supernatants from transfected cultures revealed that both SupT1 and CEM-SS cell lines supported replication of all three vif mutants to a level comparable to the parental vif-positive virus, whereas vif mutants did not replicate in H9 cells. Our results demonstrate that the requirement for Vif in SIVmac replication is cell-type dependent and that sequences near both the N and C termini are required for its function. Vif-defective SIVmac239, produced in transfected SupT1 and CEM-SS cells, failed to infect primary T lymphocytes, whereas both vif-positive and vif-defective viruses established productive infection in CEMx174 cells. These findings in primary cells suggest that Vif plays an important role in viral replication in vivo.

Transcomplementation of VIF- HIV-1 mutants in CEM cells suggests that VIF affects late steps of the viral life cycle.

The vif gene of HIV-1 has previously been claimed to be essential for the ability of cell-free virus preparations to infect cells. Here we report that the CEM T-cell-line, stably transfected with and expressing vif, supports the replication of vif- HIV-1 viruses to the same extent as wild-type HIV1. Cell entry and early replication stages are the same for vif- and vif+ HIV-1 passaged in CEM, as measured both by a PCR-based cell entry assay and by fusogenic potential. These findings indicate that vif does not affect viral infectivity on CEM cells, but seems to act at a later stage of virus replication/maturation. We also show that the VIF proteins of two different HIV-1 strains can transcomplement different vif- HIV-1 mutants.

Control of human immunodeficiency virus replication by the tat, rev, nef and protease genes.

Immediately after infection, human immunodeficiency virus directs the synthesis of three regulatory proteins tat, rev and nef that together allow the synthesis of the structural proteins of the virus after a delay of several hours. Viral mRNA production is controlled by the tat gene, which appears to stimulate elongation by RNA polymerase II, and the rev gene, which allows the accumulation of unspliced or partially spliced mRNAs in the cytoplasm. The nef gene is dispensible for virus growth but may limit virus spread by downregulating the levels of cellular surface proteins such as the CD4 receptor. Virus maturation also depends critically on the protease gene which allows the orderly rearrangement of the viral core structures in newly budded virions as well as the vpu and vif genes which allow efficient production of mature envelope glycoprotein.

Molecular biology of the human immunodeficiency virus type 1.

The immunodeficiency virus type 1 is a complex retrovirus. In addition to genes that specify the proteins of the virus particle and the replicative enzymes common to all retroviruses, HIV-1 specifies at least six additional proteins that regulate the virus life cycle. Two of these regulatory genes, tat and rev, specify proteins essential for replication. These proteins bind to specific sequences of newly synthesized virus RNA and profoundly affect virus protein expression. Tat and rev appear to be prototypes of novel eukaryotic regulatory proteins. These two genes may play a central role in regulating the rate of virus replication. Three other viral genes, vif, vpu, and vpr, affect the assembly and replication capacity of newly made virus particles. These genes may play a critical role in spread of the virus from tissue to tissue and from person to person. Our understanding of the contribution of each of the virus structural proteins and regulatory genes to the complex life cycle of the virus in natural infections is incomplete. However, enough insight has been gained into the structure and function of each of these components to provide a firm basis for rational antiviral drug development.