The replicative impairment of Vif- mutants of human immunodeficiency virus type 1 correlates with an overall defect in viral DNA synthesis.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for the infectivity of virions produced by non-permissive cells. The primary replicative defect of Vif particles involves either synthesis or stability of viral DNA, but the mechanism of this defect is unknown. Here, we report the results of a detailed analysis of HIV-1 DNA synthesis by isogenic Vif- mutants produced by different chronically infected H9 clones, which exhibit different degrees of impairment in their replicative capacity. We found that the degree of impairment of DNA synthesis by the mutant particles always correlated with the degree of their loss of infectivity. This impairment appears to be global, with a defect increasing along with synthesis of longer viral DNA species. We conclude that the primary replicative defect of Vif- virus involves the capacity of the reverse transcription complex of HIV-1 to efficiently elongate viral DNA, resulting in an inability to produce full-length viral DNA genomes.

A role for human immunodeficiency virus type 1 Vpr during infection of peripheral blood mononuclear cells.

Studies analysing human immunodeficiency virus type 1 replication in primary cells have demonstrated that Vpr, although dispensable, plays a role along with the matrix (MA) protein in allowing nuclear localization of viral preintegration complexes in non-dividing monocyte-derived macrophages (MDMs). In the current study, experimental infection conditions to analyse the role of Vpr, independently of MA, during infection of PHA/IL-2-stimulated peripheral blood mononuclear cells (PBMC) were designed. It was shown that the absence of Vpr results in a subtle effect on virus production in long-term infection. PCR analysis of the steps of virus retrotranscription during a single cycle of replication in stimulated PBMC revealed that the absence of Vpr alone correlates with an impairment in the nuclear localization of viral DNA. Our data indicate that Vpr is involved in the virus life-cycle during infection of dividing PBMC, presumably as it is during infection of MDMs.

Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor.

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes. Previous reports suggest that vif-deleted viruses are limited in replication because of a defect in the late steps of the virus life cycle. One of the remaining questions is to determine whether the functional role of Vif involves a specific interaction with virus core proteins. In this study, we demonstrate a direct interaction between Vif and the Pr55Gag precursor in vitro as well as in infected cells. No interaction is observed between Vif and the mature capsid protein. The Pr55Gag-Vif interaction is detected (i) in the glutathione S-transferase system, with in vitro-translated proteins demonstrating a critical role of the NC p7 domain of the Gag precursor; (ii) with proteins expressed in infected cells; and (iii) by coimmunoprecipitation experiments. Deletion of the C-terminal 22 amino acids of Vif abolishes its interaction with the Pr55Gag precursor. Furthermore, point mutations in the C-terminal domain of Vif which have been previously shown to abolish virus infectivity and binding to cell membranes dramatically decrease the Gag-Vif interaction. These results suggest that the interaction between Vif and the pr55Gag precursor is a critical determinant of Vif function.

Phenotypically Vif- human immunodeficiency virus type 1 is produced by chronically infected restrictive cells.

The permissivity of CD4+ transformed T cells for the replication of human immunodeficiency virus type 1 (HIV-1) vif mutants varies widely between different cell lines. Mutant vif-negative viruses propagate normally in permissive CD4+ cell lines but are unable to establish a productive infection in restrictive cell lines such as H9. As a consequence, elucidation of the function of Vif has been considerably hampered by the inherent difficulty in obtaining a stable source of authentically replication-defective vif-negative viral particles produced by restrictive cells. vif-negative, vpr-negative HIV-1 strain NDK stock, produced by the permissive SupT1 cell line, was used to infect restrictive H9 cells. By using a high multiplicity, infection of H9 cells was achieved, leading to persistent production of viral particles displaying a dramatically reduced infectious virus titer when measured in a single-cycle infectivity assay. Although these viral particles were unable to further propagate in H9 cells, they could replicate normally in CEM and SupT1 cells. Comparison of unprocessed and processed Gag proteins in the persistently produced vif-negative viral particles revealed no defect in the processing of polypeptide precursors, with no inversion of the Pr55gag/p24 ratio. In addition, there was no defect in Env incorporation for the vif-negative viral particles. Despite their apparently normal protein content, these particles were morphologically abnormal when examined by transmission electron microscopy, displaying a previously described abnormally condensed nucleoid. Chronically infected restrictive cell lines producing stable levels of phenotypically vif-negative HIV-1 particles could prove particularly useful in further studies on the function of Vif in the virus life cycle.

Requirement of caprine arthritis encephalitis virus vif gene for in vivo replication.

Replication of vif-caprine arthritis encephalitis virus (CAEV) is highly attenuated in primary goat synovial membrane cells and blood-derived macrophages compared to the wild-type (wt) virus. We investigated the requirement for CAEV Vif for in vivo replication and pathogenicity in goats by intra-articular injection of either infectious proviral DNA or viral supernatants. Wild-type CAEV DNA or virus inoculation induced persistent infection resulting in severe inflammatory arthritic lesions in the joints. We were unable to detect any sign of virus replication in vif- CAEV DNA inoculated goats, while vif- CAEV virus inoculation resulted in the seroconversion of the goats. However, virus isolation and RT-PCR analyses on blood-derived macrophage cultures remained negative throughout the experiment as well as in joint or lymphoid tissues taken at necropsy. No pathologic lesions could be observed in joint tissue sections examined at necropsy. Goats inoculated with the vif- virus demonstrated no protection against a pathogenic virus challenge. These results demonstrate that CAEV Vif is absolutely required for efficient in vivo virus replication and pathogenicity and provide additional evidence that live attenuated lentiviruses have to establish a persistent infection to induce efficient protective immunity.

The caprine arthritis encephalitis virus tat gene is dispensable for efficient viral replication in vitro and in vivo.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus closely related to visna virus and more distantly to other lentiviruses, such as human immunodeficiency virus. The genomes of visna virus and CAEV contain a tat gene encoding a protein able to weakly transactivate its own long terminal repeat, suggesting that transactivation may be a dispensable function for viral replication. Three different tat gene mutants of an infectious molecular clone of CAEV were used to study their replication after transfection or infection of primary goat synovial membrane cells and of blood-derived mononuclear cells or macrophages. Our results showed no difference between replication of the wild type and either the complete tat deletion mutant or the tat stop point mutant, whereas slower growth kinetics and lower levels of expression of the partial tat deletion mutant that of the wild type were obtained in these cells. Quantitative PCR and reverse transcription-PCR analyses of the different steps of a single replicative cycle revealed an identical pattern of retrotranscription, transcription, and viral production, whereas time course analysis demonstrated that the intracellular level of viral genomic RNA was affected by the partial tat deletion at later time points. We then compared the infectious properties of the wild-type and tat mutant viruses in vivo by direct inoculation of proviral DNAs into the joints of goats. All the animals seroconverted between 27 and 70 days postinoculation. Moreover, we were able to isolate tat mutant CAEV from blood-derived macrophages that was still able to infect synovial membrane cells in vitro. This study clearly demonstrates that the tat gene of CAEV is dispensable for viral replication in vitro and in vivo.

The vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle.

Complex retrovirus genomes contain a variable number of accessory genes, among which is the vif gene. We investigated in vitro the role of the vif gene of caprine arthritis encephalitis virus (CAEV) by studying the phenotype of five vif mutants after infection of primary goat synovial membrane (GSM) cells and blood-derived monocytes/macrophages. Any deletion introduced into the vif gene resulted in slow and low viral replication and production of virions with an infectious titer lower than that of wild-type viral particles. The wild-type phenotype could be restored by the trans expression of the vif gene in a complementation assay. Quantitative PCR and reverse transcription-PCR analyses were performed in order to determine which stage of the replicative cycle was impaired by the vif deletion. Our results demonstrated that CAEV Vif did not act at the level of reverse transcription or transcription but rather at the late stage of virus formation and/or release, as lower amounts of virus were produced after a single replicative cycle. The vif-deleted CAEV produced after 24 h of infection was still able to infect GSM cells, indicating that the vif gene is not essential for virus infectivity but is required for efficient virus production.