Replication In Primary Cells Research Articles

Inhibition of HIV-1 replication by a Tat transdominant negative mutant in human peripheral blood lymphocytes from healthy donors and HIV-1-infected patients.

It was previously shown that a tat mutant (tat22) where cysteine 22 is substituted by glycine behaves as a transdominant negative mutant in Jurkat T cells lytically or latently infected by HIV-1. In this study we demonstrate that tat22 controls HIV-1 replication in primary cells. This effect was observed both after in vitro infection of peripheral blood mononuclear cells (PBMCs) from normal donors and after reactivation of the latent infection in PBMCs from seropositive patients. The antiviral effect of tat22 was limited to conditions of low virus production. The use of tat22 may be promising for a gene therapy approach to AIDS during the asymptomatic phase of the disease allowing control of virus replication in infected cells and inhibition of virus spread to uninfected cells.

The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.

Protection of primary human T cells from HIV infection by Trev: a transdominant fusion gene.

Gene therapy is one of several approaches that are being tested in the search for an effective anti-human immunodeficiency virus (HIV) treatment. In this strategy, a "protective" gene would be introduced into target cells, rendering them relatively resistance to the virus-induced cytopathicity. Tat and Rev are viral proteins essential for HIV gene expression. Tat increases viral gene transcription and Rev is responsible for the nuclear export of mRNA encoding structural viral proteins. A fusion protein (Trev) was constructed, joining Tat and Rev transdominant mutant gene sequences. Previously, we showed that Trev inhibits both Tat and Rev activities in Jurkat T cells. To determine whether Trev could inhibit HIV replication in primary cells, we transferred the trev gene to peripheral blood lymphocytes and challenged them with different HIV strains. Levels of HIV p24 antigen (Ag) were reduced 4- to 15-fold in cultures of Trev-CD4+ T cells infected with two HIV primary clinical isolates and were not detectable in cultures infected with HIV strains NL4-3 and SF2. In contrast, cultures of nontransduced CD4+ T cells infected with the same viruses had levels of HIV p24 Ag up to 10 ng/ml. Trev-transduced CD4+ T cells demonstrated increased survival following HIV challenge for the length of the experiments (30 days). We did not observe rapid emergence of Trev-resistant HIV in our cultures. Following HIV challenge, cell-associated Trev protein was increased, supporting the hypothesis that cells surviving Trev expression provided a cell survival advantage. This work showed that Trev was able to inhibit HIV replication in primary CD4+ T cells, and, therefore the trev gene could be a candidate for gene therapy against HIV.

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.

Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild-type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.

The Carboxy-Terminal Domain Is Essential for Stability and Not for Virion Incorporation of HIV-1 Vpr into Virus Particles

Vpr is one of the auxiliary gene products encoded by HIV-1 genome. Vpr is a 14-kDa protein and exhibits several interesting characteristics including incorporation into virus particles, oligomerization, localization in the nucleus, and positive regulation of virus replication in primary cells. In an effort to define the structure–function relationship of Vpr, the role of the C-terminus of Vpr was investigated. Site-specific mutagenesis involving deletion, insertion, and substitution of residues at the C-terminus was utilized to generate variants of Vpr. Mutations introduced at the C-terminus affected properties of Vpr in different ways: (i) Vpr containing amino acids 1–72 showed the virion incorporation phenotype, indicating that the C-terminus is not essential for this function, (ii) the C-terminus contributes to the stability of Vpr, and (iii) substitution mutagenesis involving the basic residues showed stability similar to that of wild type, indicating the lack of involvement of these residues in this biochemical property of Vpr. The data generated in this study and our early mutagenic analyses on Vpr suggest that domains noncontiguous in primary sequence contribute to the stability of Vpr through overall conformation of the protein.

Effect of different donor cells on human immunodeficiency virus type 1 replication and selection in vitro.

We sought to determine the effects of different host cells on human immunodeficiency virus type 1 (HIV-1) infection in vitro. First, 17 primary viruses of various phenotypes were examined for replicative capacity in peripheral blood mononuclear cells (PBMC) from 10 healthy donors. While the range of infection was variable over a 40-fold range, it was substantially less than that previously reported (L. M. Williams and M. W. Cloyd, Virology 184:723-728, 1991). In particular, no donor cells demonstrated total resistance to HIV-1 infection. We next cocultured PBMC from an HIV-1-infected patient with stimulated PBMC from three healthy donors to determine the effect of host cells on selection for a particular HIV-1 quasispecies. By using DNA sequencing, it was found that the dominant quasispecies (AD30-15) after culture was nearly identical in the cells of different donors. Furthermore, after 6 months in vivo, the patient developed a dominant proviral population in PBMC that was most closely related to the quasispecies preferentially selected in vitro, although this quasispecies was only a minor fraction of the sequences present earlier in PBMC. In subsequent biological characterizations, it was found that AD30-15 grew much better in PBMC and macrophages than did other related quasispecies. Hence, we conclude that the primary mechanism of in vitro selection for a particular HIV-1 variant in this case is mediated by the phenotypic properties of the virus and is less dependent on host cell origin. The findings reported here have important practical implications for studies of HIV-1 replication in primary cells derived from healthy donors.

Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog.

(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50% inhibitory concentration = 0.011 to 0.057 micrograms/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.