Replication-competent Human Immunodeficiency Virus Type 1 Research Articles

Cyclophilin A-independent replication of a human immunodeficiency virus type 1 isolate carrying a small portion of the simian immunodeficiency virus SIV(MAC) gag capsid region.

Hybrid viruses between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus strain mac (SIV(MAC)) are invaluable to various fields of HIV-1 research. To date, however, no replication-competent HIV-1 strain containing the gag capsid (CA) region of SIV(MAC) has been reported. To obtain the viable gag gene chimeric virus in an HIV-1 background, seven HIV-1 strains carrying a part of SIV(MAC) CA or a small deletion in the CA region were constructed and examined for their biological and biochemical characteristics. While all the recombinants and mutants were found to express Gag and to produce progeny virions on transfection, only one chimeric virus, which has 18 bp of SIV gag CA sequence in place of the region encoding the HIV-1 CA cyclophilin A (CyPA)-binding loop, was infectious for human cell lines. Although this chimeric virus was unable to grow in monkey lymphocytic cells like wild-type (wt) HIV-1 did, it grew much better than wt virus in the presence of cyclosporin A in a human cell line which supports HIV-1 replication in a CyPA-dependent manner. These results indicate that the transfer of a small portion of the SIV(MAC) CA region to HIV-1 could confer the CyPA-independent replication potential of SIV(MAC) on the virus.

Ethanol enhances TNF-α–inducible NFκB activation and HIV-1-LTR transcription in CD4+ Jurkat T lymphocytes

During the latent phase of human immunodeficiency virus type 1 (HIV-1) infection, CD4+ T cells carrying replication-competent proviral HIV-1 DNA play an important role in persistence of the virus. Several cofactors can induce and or amplify HIV-1 replication and negatively affect disease progression and pathogenesis. Ethanol consumption is an important risk factor for HIV-1 infection, and it has been implicated in increased HIV-1 replication and progression of infection. Because tumor necrosis factor-α (TNF-α) is an important modulator of HIV-1 replication, in the present study we examined the possible effects of ethanol on TNF-α–inducible signaling associated with HIV-1 replication in human CD4+ T cells (Jurkat E6-1). We demonstrate that clinically relevant ethanol concentrations significantly potentiate TNF-α–inducible NFκB. Although ethanol effectively collaborated with TNF-α, by itself it did not have a direct effect on NFκB activation. The ethanol-dependent potentiation of TNF-α–inducible NFκB nuclear translocation was observed to involve the enhanced degradation of IκBα. Additionally, the ethanol-mediated potentiation of TNF-α–inducible NFκB activation was abrogated by the known antioxidant pyrrolidinedithiocarbamate, suggesting an important mechanistic role for reactive oxygen species in this process. In correspondence with its effect on NFκB, ethanol was also observed to significantly enhance HIV-1 long terminal repeat–dependent transcription induced by TNF-α. Overall, the data provide a molecular basis for the possible role of ethanol as a cofactor that can adversely affect HIV-1 infection and pathogenesis. (J Lab Clin Med 2000;136:333-43)

This month in J Lab Clin Med: Issue highlights for November 2000

This month in J Lab Clin Med: Issue highlights for November 2000

Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells.

Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34(+) cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.

Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals.

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.

Use of a green fluorescent protein as a marker for human immunodeficiency virus type 1 infection.

We constructed a recombinant human immunodeficiency virus type 1 (HIV-1) provirus called R7-GFP that expresses a modified form of a green fluorescent protein (GFP) from the jellyfish Aequorea victoria by substituting GFP-coding sequences for Nef-coding sequences. Alanine was substituted for serine at amino acid position 65 in the modified GFP, resulting in markedly increased fluorescence at an excitation wavelength of 488 nm as compared to wild-type GFP. The replication kinetics of R7-GFP were identical to that measured with an isogenic, nef-negative strain lacking GFP. Expression of GFP by replication-competent HIV-1 allowed simultaneous quantitation of viral infection and cell surface CD4 levels, revealing rapid and nearly complete CD4 downregulation on R7-GFP-infected PBMCs.

Analysis of vif-defective human immunodeficiency virus type 1 (HIV-1) virions synthesized in 'non-permissive' T lymphoid cells stably infected with selectable HIV-1.

In order to facilitate analyses of the molecular function of the human immunodeficiency virus type 1 (HIV-1) Vif protein, we have developed a cell culture model-system which allows permanent production of genotypically and phenotypically vif-defective HIV-1 virions in 'non-permissive' H9 cells. Using recombinant, replication-competent HIV-1 proviruses coding for a selectable marker gene (gpt) instead of nef, two stably infected H9 subclones named M2 (vif-mutant) and WX (wild-type), respectively, were generated. Virions released from cell line M2--displaying the expected vif-defective phenotype--are produced permanently, and in an at least 50 times higher amount than virus particles from acutely vif-negative HIV-1-infected H9 cells. Analysis of viral protein composition and the electron-microscopic morphology of vif-mutant virions did not reveal any detectable differences in comparison to wild-type virions.

The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures

We analyzed the leader region of human immunodeficiency virus type 1 (HIV-1) RNA to decipher the nature of the cis-acting E/psi element required for encapsidation of viral RNA into virus particles. Our data indicate that, for RNA encapsidation, there are at least two functional subregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is located between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognition signals for encapsidation. To determine whether specific putative RNA secondary structures serve as the signal(s) for encapsidation, we constructed primary base substitution mutations that would be expected to destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mutants allowed the identification of two discrete hairpins that facilitate RNA encapsidation in vivo. Thus, the HIV-1 E/psi region is a multipartite element composed of specific and functional RNA secondary structures. Compensation of the primary mutations by the second-site mutations could not be attained in trans. This indicates that interstrand base pairing between these two stem regions within the hairpins does not appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competent HIV-1 strains suggests that a subset of the hydrogen-bonded base pairs within the stems of the hairpins is likely to be required for function in cis.

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes

All presently available replication-competent proviral clones of human immunodeficiency virus type 1 (HIV-1) are derived from cell culture-amplified virus. Since tissue culture is highly selective for viral strains with an in vitro growth advantage, such clones may not be representative of the biologically relevant virus present in vivo. In this study, we report the molecular cloning and genotypic characterization of 10 HIV-1 genomes directly from uncultured brain tissue of a patient with AIDS dementia complex. Targeting unintegrated circular HIV-1 molecules for recombinant lambda phage cloning, we obtained four full-length genomes with one or two long terminal repeats (LTRs), three defective genomes with internal deletions, two rearranged genomes with inverted LTR sequences, and one integrated proviral half with flanking cellular sequences. Nucleotide sequence analysis of these clones demonstrated chromosomal integration, circle formation, genomic inversion, and LTR-mediated autointegration of HIV-1 genomes in vivo. Comparison of a 510-bp hypervariable envelope region among 8 lambda phage-derived and 12 polymerase chain reaction-derived clones from the same brain specimen identified a predominant viral form as well as genetically divergent variants. Variability among 19 of 20 clones ranged between 0.2 and 1.2%. One clone exhibited 8.2% nucleotide sequence differences consisting almost exclusively of G-to-A changes. Transfection of the four full-length HIV-1 genomes identified one clone (YU-2) as replication competent and exhibiting growth characteristics similar to those of tissue culture-derived macrophage tropic strains of HIV-1. These results demonstrate, for the first time, that replication-competent HIV-1 genomes, complex mixtures of defective viral forms, and chromosomally integrated provirus persist in vivo. In addition, the brain-derived viral clones are expected to prove valuable for future studies of macrophage and neurotropism as well as for the analysis of other viral properties that are subject to in vitro selection pressures.

Absence of Infectious HIV-1 in the Urine of Seropositive Viremic Subjects

Urine and peripheral blood samples from 48 human immunodeficiency virus type 1 (HIV-1) seropositive individuals (38 adults and 10 children) were evaluated for the presence of HIV-1 by cocultivation and for HIV-1 p24 antigen by ELISA. None of the urine samples contained replication-competent HIV-1; 41 (85%) of 48 simultaneously obtained peripheral blood mononuclear cell samples contained replication-competent HIV-1. None of 26 urine samples available for analysis contained HIV-1 p24 antigen as determined by ELISA; 12 (34%) of 35 simultaneously obtained peripheral blood samples had detectable serum HIV-1 p24 antigen. Two of the individuals studied had HIV nephropathy, three had pyuria, and five had microscopic hematuria. Culture sensitivity was maximal when mycostatin (and not amphotericin B) was used as an antifungal agent. Our findings indicate that urine from HIV-1-seropositive individuals is unlikely to contain infectious HIV-1. This would imply that the risk of transmission of HIV-1 by urine is low to nonexistent.