High frequency of defective vpu compared with tat and rev genes in brain from patients with HIV type 1-associated dementia.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated dementia (HAD) and other neurological disorders. The role of HIV regulatory and accessory proteins in the pathogenesis of these disorders is unclear. Here we analyzed sequences of tat, rev, and vpu genes in 55 subgenomic clones previously shown to encode functional env genes from brain and lymphoid tissues of four AIDS patients with HAD. Phylogenetic analysis showed distinct compartmentalization of tat, rev, and vpu genes in brain versus lymphoid tissues. Nine of 19 vpu sequences from brain of two patients had premature stop codons at positions between amino acids 2 and 30, compared with 0 of 8 from lymphoid tissues. Tat sequences from brain (n = 8 of 8) but not lymphoid (n = 0 of 6) tissue from one patient had a 35 amino acid truncation at the C-terminus. Rev sequences from the brain of one patient (n = 6 of 8) had a 5 amino acid truncation. These results demonstrate a high frequency of defective vpu compared with tat and rev genes in brain from HAD patients, and identify sequence variants of these regulatory/accessory genes that may influence the pathogenesis of HIV-associated neurological disease.

Construction of a minimal HIV-1 variant that selectively replicates in leukemic derived T-cell lines: towards a new virotherapy approach.

T-cell acute lymphoblastic leukemia is a high-risk type of blood-cell cancer. We analyzed the possibility of developing virotherapy for T-cell acute lymphoblastic leukemia. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. We constructed a minimized derivative of HIV-1, a complex lentivirus encoding multiple accessory functions that are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. This mini-HIV virus has five deletions (vif, vpR, vpU, nef, and U3) and replicated in the SupT1 cell line, but did not replicate in normal peripheral blood mononuclear cells. The stripped down mini-HIV variant was also able to efficiently remove leukemic cells from a mixed culture with untransformed control cells. In contrast to wild-type HIV-1, we did not observe bystander killing in mixed culture experiments with the mini-HIV variant. Furthermore, viral escape was not detected in long-term cultures. The mini-HIV variant that uses CD4 and CXCR4 for cell entry could potentially be used against CXCR4-expressing malignancies such as T-lymphoblastic leukemia/lymphoma, natural killer leukemia, and some myeloid leukemias.

Characterization of pol, vif, vpr, and vpu genes of HIV type 1 in AIDS patients with high viral load and stable CD4+ T cell counts on combination therapy.

The success of combination therapy has also led to AIDS patients who exhibit elevated viral load without a corresponding decline in CD4+ T cells. In this study, we characterized changes in the pol gene and accessory genes vif, vpr, and vpu of HIV-1 isolated from the plasma of patients receiving highly active antiretroviral therapy. From each patient three sequences were obtained and compared with the sequence of HIV-1 from nontreated patients, revealing many substitutions that were similar in most cases. Protease and reverse transcriptase genes showed many mutations that were due to antiviral drugs. Premature termination was observed in the vif gene of one patient, leading to a protein truncated after 187 amino acids. In another patient the entire vpr open reading frame was missing, with no synthesis of Vpu protein because the 5' end of the gene was missing, including the start codon. In the same patient, the Vif protein was also truncated because of the deletion of 100 nucleotides at the 3' end of the vif gene.

Characterization and phylogenetic analysis of South African HIV-1 subtype C accessory genes.

To acquire new knowledge about the genetic diversity and potential impact on vaccine strategies of HIV-1 subtype C in South Africa, we have characterized the vif, vpr, and vpu genes of 15 isolates. Phylogenetic analysis of the genomic fragment encompassing these genes revealed subtype C subclusters, suggesting close relatedness with subtype C strains from other geographic locations and excluded isolation of South African strains. The putative T155 phosphorylation site in the C terminal of Vif was absent in all subtype C sequences. Variation in the predicted amino acid sequences of the three genes further showed strong correlation with other subtype C sequences.

[Mechanisms of reduction of CD4 receptor expression on the surface of HIV-1 infected cells]. / Mécanismes de réduction de l'expression des récepteurs CD4 à la surface des cellules infectées par le VIH-1.

Specific interactions between the cell surface CD4 receptor and the HIV-1 envelope glycoprotein gp120 are responsible for the entry of HIV into host cells. Following infection, a down-modulation of CD4 at the cell surface is commonly observed. This may render cells resistant to subsequent infection by HIV as well as other viruses that also use CD4 as a portal of entry. This phenomenon is termed retroviral interference. CD4 down-modulation is complex and involves at least 3 viral gene products which include the envelope precursor gp160 and 2 auxilliary proteins Nef and Vpu. CD4 down-modulation has been observed in each of primary CD4+ T-lymphocytes and monocyte-derived macrophages, as well as both T and monocytic cell lines. CD4 down-regulation may occur at different levels. Specific binding of soluble gp120 may lead to internalization of CD4. The HIV-1 nef gene product which is expressed prior to HIV-1 structural proteins also causes the internalization of CD4 followed by its lysosomal degradation. During the late phase of viral gene expression i.e. viral structural protein synthesis, CD4-gp160 complexes forming in the ER represent another important factor leading to CD4 down-modulation. Finally, CD4 which is retained by gp160 in the ER, is specifically degraded in the presence of Vpu. Thus, it appears that CD4 down-regulation is of central importance to the life cycle of HIV-1.

vpu and env sequence variability of HIV-1 isolates from Tanzania.

PIP: The nucleotide sequences of an approximately 1400-base pair (bp) region spanning the vpu and env (V1-V3) genes of 9 HIV-1 isolates originating from Tanzania were determined. Peripheral blood lymphocyte (PBL) specimens were obtained in 1988 from urban patients with clinical signs of AIDS attending the Muhimbili Medical Center, Dar es Salaam, Tanzania. 9 samples (TZ005, TZ012, TZ016, TZ017, TZ023, TZ030, TZ053, TZ064, and TZ112) were randomly chosen for virus isolation by cocultivation with HIV-negative donor PBLs. Viral DNA sequences between the positions 5543 and 6956 were amplified by polymerase chain reaction (PCR), using 2 sets of primer pairs, subcloned into a Bluescript vector, and sequenced on both strands. Sequence analysis revealed vpu and env open reading frames (ORFs) for all clones, except 2 that had a missense mutation in vpu (TZ016) or env (TZ017). The vpu sequences showed a high degree of homology among all isolates, with TZ005, TZ016, and TZ030 having identical sequences. Phylogenetic tree analysis indicated that most of the isolates fell into the D subtype. The analysis of the deduced protein sequences of the V3 loop, which contains the principal neutralizing domain (PND), revealed an amino acid pattern closely related, but not identical, to known African HIV isolates. The GSGQ motif was found in 4 isolates (TZ005, TZ030, TZ053, and TZ080), and the GPGQ motif was found in 2 cases (TZ016 and TZ017). The V3 variability of the HIV isolates was greater than previously reported for Tanzanian viruses. Although AIDS viruses are believed to have originated from Africa, little is known about the sequence variability of African HIV-1 isolates, compared to the information available on Euro-American viruses. The variability of East African HIV-1 isolates are consistent with the view that these are rapidly changing viruses for which further variants are likely to be discovered.^ieng

Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate.

Macrophages and lymphocytes are the two main targets for productive HIV-1 infection in vivo. To compare the effects of the "nonessential" HIV-1 accessory genes vpr, vpu, and nef on viral replication in these primary cell types, we generated a panel of mutant viruses derived from a molecularly cloned macrophage-tropic HIV-1 primary isolate. Mutant viruses had markedly different patterns of replication in macrophages, in contrast to lymphocytes in which differences were modest. Loss of vpr or vpu reduced viral antigen production in macrophages by up to 1000-fold, while replication in lymphocytes was only marginally affected. Loss of nef did not affect lymphocyte infection, but decreased replication in macrophages to a small extent. Mutation of multiple accessory genes restricted replication in both cell types, but to a much greater extent in macrophages, and frequently resulted in nonproductive infection. The degree to which replication depended on intact accessory genes varied in macrophages from different donors. The essential functions of these accessory genes in HIV-1 infection may be related to their combined effects in facilitating productive infection of macrophages.

Integration is essential for efficient gene expression of human immunodeficiency virus type 1.

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus.

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.

High-resolution structure of an HIV-1 quasispecies: identification of novel coding sequences.

OBJECTIVE: To characterize an HIV-1 quasispecies in vivo at high resolution (1%) in order to determine its genetic structure. METHODS: The first coding exon of tat was amplified by polymerase chain reaction from uncultured peripheral blood mononuclear cells of an HIV-1-infected patient. The products were cloned into M13mp18 RF, and 106 clones were sequenced. RESULTS: Thirty-one different Tat protein variants were found. Amongst these, five major forms with frequencies of 44, 11, 8 and 5% were identified. All of the remaining 26 sequences were unique, 15 of which were defective. Within the variant spectrum a small number of genomes encoded novel open reading frames, for example, a tat-vpu fusion product. CONCLUSION: Some of the myriad proviruses present in an individual harbour novel coding sequences. While these are probably of little importance for AIDS pathogenesis they emphasize the ability of HIV to explore a huge range of genetic configurations.

Dual regulation of silent and productive infection in monocytes by distinct human immunodeficiency virus type 1 determinants.

The regulation of human immunodeficiency virus type 1 infection and replication in primary monocytes was investigated by mutagenesis of recombinant proviral clones containing an env determinant required for the infectivity of monocytes. Virus replication was assayed by determination of reverse transcriptase activity in culture fluids and by recovery of virus from monocytes following cocultivation with uninfected peripheral blood mononuclear cells. Three virus replication phenotypes were observed in monocytes: productive infection, silent infection, and no infection. Incorporation of the monocytetropic env determinant in a full-length clone incapable of infection or replication in primary monocytes (no infection) conferred the capacity for highly efficient virus replication in monocytes (productive infection). Clones with the env determinant but lacking either functional vpr or vpu genes generated lower replication levels in monocytes. Mutation of both vpr and vpu, however, resulted in nearly complete attenuation of virus replication in monocytes, despite subsequent virus recovery from infected monocytes by cocultivation with uninfected peripheral blood mononuclear cells (silent infection). These findings indicate a central role for the "accessory" genes vpu and vpr in productive human immunodeficiency virus type 1 replication in monocytes and indicate that vpu and vpr may be capable of functional complementation.

Cells surviving infection by human immunodeficiency virus type 1: vif or vpu mutants produce non-infectious or markedly less cytopathic viruses.

Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non-infectious or less cytopathic virus.