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At least 19 recordsLinked to original sources
PubMed2005

The middle to 3' end of the HIV-1 vif gene sequence is important for vif biological activity and could be used for antisense oligonucleotide targets.

The human immunodeficiency virus type-1 (HIV-1)-encoded vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 Vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its encapsidation into virions. Some studies have established targets within the HIV-1 vif gene that are important for its biologic function; however, it is important to determine effective therapeutic targets in vif because of its critical role in HIV-1 infectivity and pathogenicity. The present study demonstrates that virions generated in transfected HeLa-CD4+ cells, especially from HIV-1 vif frame-shift mutant (3' delta vif; 5561-5849), were affected in splicing and had low infectivity in MT-4 cells. In addition, HIV-1 vif antisense RNA fragments constructed within the same region, notably the region spanning nucleic acid positions 5561-5705 (M-3'-AS), which corresponds to amino acid residues 96-144, significantly inhibited HIV-1 replication in MT-4 and reduced the HIV-1 vif mRNA transcripts and reporter gene (EGFP) expression. The generated virions showed low secondary infection in H9 cells. These data therefore suggest that the middle to the 3' end of vif is important for its biological activity in the target cells.

3' Flanking Region↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Analysis of the vif gene of feline immunodeficiency virus.

We have examined FIV vif function in the context of molecular clone pF34 (GenBank Accession No. M25729). Rabbit antisera directed against the translation product of the vif gene identified a 29-kDa protein in tissue culture cells infected with FIV-pF34; this protein was not present in cultures of uninfected cells. Thus, the vif gene of this virus strain is expressed in infected cells. Three mutations were made in distinct regions of the vif gene of molecular clone FIV-pF34: (i) deletion of 223 bp from the central portion of the gene, (ii) site-directed mutation of a conserved N-terminal basic region, and (iii) site-directed mutation of a conserved C-terminal motif. FIV proviruses containing each of these mutations were assessed for replication following transfection into two feline adherent cell types, CrFK and G355-5. Reverse transcriptase and p24gag antigen assays of supernatants from transfected cultures revealed that all three vif mutants produced very little cell-free virus or viral protein in both cell types. Results of immunocytochemical staining of these cultures indicated that all three mutants expressed low levels of cell-associated FIV p24gag. These findings suggest that each of the three regions mutated in vif is critical for function. Our observations are consistent with studies showing marked attenuation of HIV-1 vif mutants in certain cell types. We conclude that the vif gene is a critical determinant of FIV-pF34 replication and infectivity in CrFK and G355-5 cells.

Amino Acid Sequence↗
PubMed1994

In vivo genetic variability of the HIV-1 vif gene.

The human immunodeficiency virus type 1 (HIV-1) vif gene encodes a 23-kDa protein (viral infectivity factor) whose exact mechanism of action is not entirely clear. Vif is believed to be highly conserved among different HIV-1 strains. We have analyzed the proviral vif sequences of 61 peripheral blood mononuclear cell samples from HIV-1 positive patients by direct solid phase sequencing and temperature gradient gel electrophoresis of polymerase chain reaction products. Inter- and intraindividual sequence variations, conserved motifs, and prevalent vif subtypes were investigated. The consensus proviral vif DNA sequence of the 61 samples as well as the consensus sequence of the 61 deduced vif amino acid sequences were found to be less conserved than previously thought. The vif proviral sequences were 58% conserved, with the 5' end of the vif gene being the most conserved region (84%). Of the vif amino acids only 45% were absolutely conserved in the 61 samples, i.e., the absolutely conserved and as such possibly functionally important domains of the vif protein comprised less than half of the vif amino acids. In two-thirds of the variable positions residues belonging to different amino acid groups were found. In individual patients the prevalent vif sequence changed with the course of disease, but the differences found in two serial samples of a patient were < or = 10%. Phylogenetic analysis revealed that one African vif subtype had been introduced in the investigated population.

Amino Acid Sequence↗
PubMed1997

Diversity of the vif gene of human immunodeficiency virus type 1 in Uganda.

Little sequence information is available for human immunodeficiency virus type 1 (HIV-1) vif genes of African origin. Here we describe 37 new complete vif genes of 18 AIDS patients from Uganda and show that vif has a high in vivo genetic variability. vif proviral DNA sequences of peripheral blood cells were determined by direct sequencing of PCR products. Only 52% of the deduced Vif amino acids were absolutely conserved; when Vif sequences previously analysed were considered, only 32% of the Vif consensus sequence comprised conserved and as such possibly functionally important motifs. The high inter-individual vif variability was in contrast to a very low intra-individual variability. One patient carried a vif gene with a stable C-terminal deletion, but N-terminal truncations were not found in patients' predominant vif sequences. The vif genes analysed comprised subtypes A, D and an A/D mosaic. Phylogenetic analyses additionally showed that HIV- 1 in Uganda has spread across the boundaries of ethnic groups.

Acquired Immunodeficiency Syndrome↗
PubMed1996

A high frequency of defective vif genes in peripheral blood mononuclear cells from HIV type 1-infected individuals.

We studied the heterogeneity and intactness of the vif gene in six HIV-1-infected individuals at various clinical stages. The proviral vif sequences in peripheral blood mononuclear cells were amplified by PCR, followed by cloning and sequencing of 45 vif clones. The intraindividual diversity of the vif genes ranged from 0.45 to 3.3% and was not correlated with disease stage. Although the vif gene has been shown to be essential for infection of HIV-1 in vitro, a high frequency (31%) of defective vif genes was observed. In one patient, six vif clones carried double nonsense mutations at the same positions, five of which were clustered in the phylogenetic tree, suggesting that these vif-defective viruses may have replicated in vivo. Phylogenetic analysis revealed that the vif sequences from each individual were clustered into a separate group and that all of them belong to subtype B.

Adult↗
PubMed1998

Conservation of an intact vif gene of human immunodeficiency virus type 1 during maternal-fetal transmission.

The human immunodeficiency virus type 1 (HIV-1) vif gene is conserved among most lentiviruses, suggesting that vif is important for natural infection. To determine whether an intact vif gene is positively selected during mother-to-infant transmission, we analyzed vif sequences from five infected mother-infant pairs following perinatal transmission. The coding potential of the vif open reading frame directly derived from uncultured peripheral blood mononuclear cell DNA was maintained in most of the 78,912 bp sequenced. We found that 123 of the 137 clones analyzed showed an 89.8% frequency of intact vif open reading frames. There was a low degree of heterogeneity of vif genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vif sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Furthermore, the epidemiologically linked mother-infant pair vif sequences displayed similar patterns that were not seen in vif sequences from epidemiologically unlinked individuals. The functional domains, including the two cysteines at positions 114 and 133, a serine phosphorylation site at position 144, and the C-terminal basic amino acids essential for vif protein function, were highly conserved in most of the sequences. Phylogenetic analyses of 137 mother-infant pair vif sequences and 187 other available vif sequences from HIV-1 databases revealed distinct clusters for vif sequences from each mother-infant pair and for other vif sequences. Taken together, these findings suggest that vif plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.

Adult↗
PubMed1999

Mutational analysis of human immunodeficiency virus type 1 vif gene.

Mutations were introduced into scattered regions of the HIV-1 vif gene. The twelve in-frame mutants generated were evaluated for the replication potentials in cells by transfection and infection experiments. All the mutants produced a normal level of progeny virions upon transfection, indicating the absence of the late function of HIV-1 Vif protein. The infectivity of virions obtained was monitored in H9 cells, which are non-permissive for HIV-1 without the Vif function. Most of the mutations in various parts of the vif gene, including those in the three conserved regions among HIV/SIV, abrogated the infectivity of the virus. In contrast, the cysteine residue at position 133, which was reported to be critical for viral infectivity, was found not to be essential. In addition, the C-terminal eight amino acid residues (185-192) in the Vif protein could be deleted with no effects on viral growth potential.

Gene Products, vif↗
PubMed1996

Unimpaired function of a naturally occurring C terminally truncated vif gene product of human immunodeficiency virus type 1.

In approximate 10 percent of natural human immunodeficiency virus 1 (HIV-1) vif gene populations, sequences of shortened vif open reading frames with premature stop codons have been found. Here we report the functional analysis of two patient-derived vif genes. Vif45-2 encodes a C terminally truncated Vif protein of only 173 instead of 192 amino acids and additionally contains several rare amino acid substitutions which are in part shared by vifA65-5. HIV-1 pNL4-3-derived recombinant A45-2 and A65-5 virions were fully infectious in H9 cells and human PBMC, both known to be non-permissive for vif-defective HIV-1. Furthermore, A45-2 virions produced in primary human monocyte-derived macrophages were infectious for MT-4 cells. This study unequivocally demonstrates that the C-terminal region (19 amino acids) of the Vif protein is dispensable for Vif function in the in vitro cell culture systems employed. Additionally, we investigated whether the Vif protein might be phosphorylated in vivo and obtained no evidence for this.

Amino Acid Sequence↗
PubMed1999

The feline immunodeficiency virus vif gene is required for productive infection of feline peripheral blood mononuclear cells and monocyte-derived macrophages.

The role of the feline immunodeficiency virus (FIV) vif gene in establishing productive infection in feline peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) was examined in cell culture systems. A 375-bp deletion was introduced into the vif gene of the wild-type FIV-pPPR infectious molecular clone to produce Vif deletion mutant FIV-pPPRDeltavif. This mutant FIV proviral construct expressed FIV proteins p24gag and gp100env in transfected Crandell feline kidney cells as measured by immunoprecipitation and Western blot analysis as well as immunocytochemical analysis; these cultures produced very low levels of virus by cocultivation of transfected cells with PBMCs and K-258 cells, as measured by production of p24gag. Replication kinetics of wild-type and vif-deleted virus were compared in PBMCs and monocyte-derived macrophages (MDMs) by infection with cell-free virus preparations. Similar to findings with other lentiviruses, the vif gene was found to be essential for establishment of productive FIV infection in both PBMCs and MDMs. This study indicates that vif is essential for productive FIV infection of host target cells in vitro and that FIV-pPPRDeltavif may be an excellent candidate viral mutant for attenuated virus vaccine studies.

Animals↗
PubMed2004

Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression.

Two HIV-1 accessory proteins, Vpu and Vif, are notoriously difficult to express autonomously in the absence of the viral Tat and Rev proteins. We examined whether the codon bias observed in the vpu and vif genes relative to highly expressed human genes contributes to the Rev dependence and low expression level outside the context of the viral genome. The entire vpu gene as well as the 5' half of the vif gene were codon optimized and the resulting open reading frames (ORFs) (vphu and hvif, respectively) were cloned in autonomous expression vectors under the transcriptional control of the CMV promoter. Codon optimization efficiently removed the expression block observed in the native genes and allowed high levels of Rev- and Tat-independent expression of Vpu and Vif. Most of the higher protein levels detected are accounted for by enhanced steady-state levels of the mRNA encoding the optimized species. Nuclear run-on experiments show for the first time that codon optimization has no effect on the rate of transcriptional initiation or elongation of the vphu mRNA. Instead, optimization of the vpu gene was found to stabilize the vphu mRNA in the nucleus and enhance its export to the cytoplasm. This was achieved by allowing the optimized mRNA to use a new CRM I-independent nuclear export pathway. This work provides a better understanding of the molecular mechanisms underlying the process of codon optimization and introduces novel tools to study the biological functions of the Vpu and Vif proteins independently of other viral proteins.

Cell Line↗
PubMed2004

Intracellular expression of antisense RNA transcripts complementary to the human immunodeficiency virus type-1 vif gene inhibits viral replication in infected T-lymphoblastoid cells.

The human immunodeficiency virus type-1 (HIV-1)-encoded vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its virions from encapsidation. Although some studies have established targets within the HIV-1 vif gene that are important for its biologic function, it is however important to further screen for effective therapeutic targets in the vif gene that could interfere with the HIV-1 vif-dependent infectivity and pathogenicity. This report demonstrates that HIV-1 vif antisense RNA fragments constructed within mid-3' region, notably the region spanning nucleic acid positions 5561-5705 (M-3'-AS), significantly inhibited HIV-1 replication in MT-4 and H9-infected cells and reduced the HIV-1 vif mRNA transcripts. These data clearly suggest that the above vif fragment, which corresponds to amino acid residues 96-144, could be an effective novel therapeutic target site for gene therapy applications, for the control and management of HIV-1 infection, due to its strong inhibition of HIV-1 replication in cells.

Base Sequence↗
PubMed2006

Molecular phylogenetic analysis of HIV-1 vif gene from Korean isolates.

Phylogenetic studies of nef, pol, and env gene sequences of HIV-1 isolated from Koreans suggested the presence of a Korean clade in which Korean sequences are clustered to the exclusion of foreign sequences. We attempted to identify and characterize the Korean clade using all vif gene sequences isolated from Koreans registered in the NCBI GenBank database (n = 233). Most (77 %) of the Korean isolates belonged to the Korean clade as a large subcluster in subtype B, designated the Korean clade subtype B (KCB). KCB sequences were relatively homogenous compared to Korean subtype B sequences that did not belong to the KCB (non-Korean clade subtype B; NKCB). Comparison of amino acid frequencies of KCB and NKCB sequences revealed several positions where the amino acid frequencies were significantly different. These amino acid residues were critical in separating KCB from NKCB or from foreign sequences, since substitution of these amino acids in KCB with the NKCB amino acids relocated the KCB sequences to NKCB, and vice versa. Further analyses of KCB will help us to understand the origin and evolutionary history of KCB.

Amino Acid Sequence↗
PubMed1995

Conservation of an intact human immunodeficiency virus type 1 vif gene in vitro and in vivo.

Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.

Amino Acid Sequence↗
PubMed1995

Sequence analysis of HIV-1 vif gene in Spanish isolates.

We have determined the nucleotide sequence of the HIV-1 vif gene in viruses obtained from symptomatic patients of distinct risk groups in Madrid. The genetic diversity among the isolates was estimated in 4.6% (+/- 1.4 standard deviation), a similar value to that obtained for the gag gene 3.9% (+/- 0.8 standard deviation) and env 4.1% (+/- 1 standard deviation) (Rojas et al., Virus Res 31, 331-342, 1994). Amino acid sequence analysis revealed the presence of hypermutable residues at positions 101 and 167, close to antigenically relevant sequential epitopes (comprising amino acids 87-94 and 172-178). Phylogenetic analysis supports the existence of two virus lineages circulating preferentially within different risk groups.

Adult↗
PubMedDate not supplied

[Expression of the vif gene of human immunodeficiency virus type 1 in Escherichia coli and study of the immunoreactivity of the vif protein].

Full-sized gen vif of human immunodeficiency virus has been synthesized and cloned into plasmid pGEX-2T. Vif-gene expression was found in Escherichia coli cells resulting in production of a hybrid GST-protein. The recombinant protein studied by the immunoblotting technique reacted with 8 of 22 probes of human HIV-positive sera. The recombinant protein is specifically cut by thrombin in two proteins corresponding to GST and VIF-proteins in molecular mass.

Base Sequence↗
PubMed2000

Characterization of human immunodeficiency virus type 1 vif gene in long-term asymptomatic individuals.

We have determined the sequence of the human immunodeficiency virus type 1 (HIV-1) vif genes from a cohort of 42 long-term nonprogressors (LTNP) and compared these sequences to those of 8 late progressors. The coding potential of the vif open reading frame directly derived by nested PCR from uncultured peripheral blood mononuclear cell DNA was conserved in all 50 individuals. The nucleotide distances between vif sequences were not significantly different between LTNP and late progressors, indicating similar selections of viruses within both types of long-term HIV-1-infected subjects. However, a statistically significant correlation between an amino acid signature at position 132 of Vif and the viral load was found within LTNP. Namely, amino acid Ser was associated with low viral load and amino acid Arg with high viral load. This signature was also observed when LTNP with low viral load were compared to progressors. The Ser132 signature was introduced in place of Arg132 present in the HIV-1 YU-2 Vif prototype into chimeric viruses to assess the impact of Vif signature on the virus. While the replication properties in the SupT1 cell line were unmodified, the mutagenized virus revealed a fivefold decreased replication in activated PBMC, suggesting a possible role of this Vif signature for viral production in vivo.

Acquired Immunodeficiency Syndrome↗