Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.     

Phagocytosis reduces HIV-1 production in human monocytes/macrophages infected in vitro

Summary The addition of ingestible particles (opsonized erythrocytes or latex beads) or a phorbol ester activates monocytes — derived human macrophages (MDHM) cultured in vitro, and markedly reduces virion release from HIV-infected MDHM as well as their ability to transmit the infection to cocultured lymphoid CD 4-positive CEM cells.     

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.     

Severe herpetic whitlow in an HIV-1 and HIV-2 infected patient

Our paper describes an unusual case of herpetic whitlow due to HSV-2 in an HIV-1 and HIV-2 infected patient. This patient was a 33-year-old cook, HIV-1Ab and HIV-2Ab positive for 4 years. The CD4+ cell count was below 50 cells/microL and no previous AIDS-defining illness happened. After having had a jagged tearing wound by a carving-knife on index finger of his right hand, he showed a rapid advancing erosion, which completely encircled his forefinger, due to HSV-2. Twenty days later he also showed two small adjacent lesions on penile shaft which rapidly extended with multiple subpreputial lesions. These lesions were caused by HSV-2 infection too. Both, finger and penile lesions, completely healed after a 3-week treatment with intravenous and oral acyclovir.     

The budding of defective human immunodeficiency virus type 1 (HIV-1) particles from cell clones persistently infected with HIV-1

Summary Three cell clones producing large numbers of infectious or noninfectious particles of human immunodeficiency virus type 1 (HIV-1), designated M 10/LAV-2, M 16/LAV-3, and MT/LAV-17, were isolated from persistently HIV-1-infected MT-4 cells. In M 10/LAV-2, the HIV-1 proteins were defective in the cleavage ofgag precursor protein, and the particles were doughnutshaped with a double-ring structure. These particles were produced by budding at the cell surface from crescentic structures followed by the formation of double-ring structures. The viral proteins in M 16/LAV-3 were defective in the cleavage ofenv precursor protein. The morphology of the virus particles was intact, and an electron dense bar-shaped core was seen inside a single-ring enveloped structure. The intact particles were released from the cell surface by a budding process in which crescent shape structures first appeared at the cell membrane, then subsequently just before release matured to a complete structure with an electron dense core. In MT/LAV-17, the synthesis of HIV-1 proteins was normal, and the particles were teardrop-shaped with an intact core structure. These particles were produced by budding with an electron dense core at the cell surface.Thus, it was suggested that the morphological maturation of HIV-1 particles was completed just before release from the cell surface in several cell clones producing HIV-1 particles of different morphology.     

HIV-1 Env-chemokine receptor interactions in primary human macrophages: entry and beyond

While HIV has subverted the chemokine receptors CCR5 and CXCR4 for its own use as an entry co-receptor, their normal functions are to transduce signals in response to extracellular ligands. Our lab is interested in understanding how HIV-1 glycoprotein 120 (gp120) may activate intracellular signals through these receptors in primary human macrophages, and how these responses may contribute to pathogenesis. Our studies demonstrate HIV-1 gp120 elicits several different types of signals in macrophages through CXCR4 and CCR5, including calcium elevations, ionic channel activation, non-receptor protein tyrosine kinase activation, and activation of MAP kinases. Receptor activation is triggered by both monomeric gp120 and whole HIV virus. Furthermore, gp120 elicits a number of functional responses in macrophages, such as secretion of chemokines and other soluble products, and we demonstrate that specific pathways linked to the chemokine receptors are responsible. These studies help illuminate the pathways through which chemokine receptors are coupled in primary macrophages, and provide a mechanistic basis for effects that HIV has on macrophage function. These signaling responses may play a role in the pathogenesis of organ dysfunction such as HIV encephalopathy and lymphocytic interstitial pneumonitis where macrophages are the principal infected cell type and inappropriate immune activation plays a central role.     

The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages

Binding of HIV-1 envelope glycoprotein (Env) to its cellular receptors elicits a variety of signaling events, including the activation of select tyrosine kinases. To evaluate the potential role of such signaling, we examined the effects of the tyrosine kinase inhibitor, genistein, on HIV-1 entry and infection of human macrophages using a variety of assays. Without altering cell viability, cell surface expression of CD4 and CCR5 or their abilities to interact with Env, genistein inhibited infection of macrophages by reporter gene-encoding, beta-lactamase containing, or wild type virions, as well as Env-mediated cell-fusion. The observation that genistein blocked virus infection if applied before, during or immediately after the infection period, but not 24h later; coupled with a more pronounced inhibition of infection in the reporter gene assays as compared to both beta-lactamase and p24 particle entry assays, imply that genistein exerts its inhibitory effects on both entry and early post-entry steps. These findings suggest that other exploitable targets, or steps, of the HIV-1 infection process may exist and could serve as additional opportunities for the development of new therapeutics.     

Identification of HLA-A*3101-restricted cytotoxic T-lymphocyte response to human immunodeficiency virus type 1 (HIV-1) in patients with chronic HIV-1 infection

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infections. As several HIV-1 CTL epitopes restricted to many HLA types are already known, we aimed at identifying the CTL epitopes restricted by HLA-A*3101 in an effort to expand the epitope repertoire available for the development of potential T cell-mediated therapeutic measures and protective vaccines. Scanning of HIV-1 clade B SF2 strain proteins for the presence of peptides containing HLA-A*3101-binding motifs revealed 88 nine- to 11-mer peptides that had been synthesized and assayed for binding to HLA-A*3101 molecules. Peptides with medium to high HLA-binding affinity were tested for their ability to stimulate a CTL response in the peripheral blood mononuclear cells (PBMCs) from selected HIV-1-infected patients. Two of these binding peptides, Env769-779 (RLRDLLLIAAR) and Nef192-200 (KLAFHHMAR), induced peptide-specific CTLs in PBMCs from at least two of five HIV-1-seropositive individuals. CTL clones specific for the two peptides killed HLA-A*3101-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed HLA-A*3101-restricted CTL epitopes. Identification of T-cell epitopes on HIV-1 proteins will increase our understanding of the role of CD8+ T cells in HIV-1 infections and assist in the design of new protective strategies.     

Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes.

HIV-1 NEF genes were isolated directly from peripheral blood lymphocyte DNA of two HIV-1-infected individuals and cloned into an HXB-2-infectious molecular clone. The effect of NEF on virus production in T-cell lines and primary human lymphocytes was studied. Naturally occurring NEF accelerates virus production in primary human lymphocytes, but not in T-cell lines.     

HIV-1 extrachromosomal 2-LTR circular DNA is long-lived in human macrophages

HIV-1 extrachromosomal 2-LTR circles (cc2LTR) are rapidly lost in dividing cell populations and, therefore, might be interpreted as representing new infection and ongoing viral replication. However, recent work demonstrated that cc2LTR persist in infected, growth-arrested T cell lines beyond their predicted half-life as previously determined in dividing cell populations. In this study, the evaluation of the stability of cc2LTR was extended to include primary human macrophages, a natural, non-dividing target of HIV-1. By quantitative real-time PCR, cc2LTR were found to persist out to 21 days post-infection in macrophages infected with both integrase competent and integrase- defective, recombinant HIV-1, whereas in activated CD4(+) T lymphocytes, they rapidly decreased over time. This persistence was associated with persistent, low level expression of the indicator gene, luciferase. These data suggest that the presence of HIV-1 cc2LTR in the PBMC of HIV-1-infected patients on suppressive HAART could be due either to ongoing generation of newly infected dividing cells, or persistence of circles in non-dividing cell populations where they appear to be stable. Furthermore, exrachromosomal circular DNA in this cell population could be a source of persisent viral protein expression.     

Fine serotyping of human immunodeficiency virus serotype 1 (HIV-1) and HIV-2 infections by using synthetic oligopeptides representing an immunodominant domain of HIV-1 and HIV-2/simian immunodeficiency virus.

In this study, enzyme immunoassays for detection of type-specific antibodies to human immunodeficiency viruses (HIV) were developed by using short peptides corresponding to sequences located within the immunodominant domain of the transmembrane glycoproteins of both HIV-1 and HIV-2-simian immunodeficiency virus (SIV). The assays were highly sensitive with currently available sera from various geographical areas. Furthermore, they appeared to be more specific in HIV serotyping than the Western blot (immunoblot) assay, since all of the sera were clearly discriminated as one or the other type. It was also shown that in contrast to HIV-1, the C-terminal cysteine residue (amino acid 620, SIV from captive macaques, Mm142 strain) of the HIV-2-SIV peptide is not necessary for recognition of the peptide by antibody to HIV-2.     

Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes

HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins. When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins. Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL. Preformed HLA-peptide complexes may provide an alternative to immunization procedures that depend upon intracellular processing of antigen to elicit T cell responses.      

Differentiation of human immunodeficiency virus type 1 (HIV-1) infections with HIV-2-cross-reacting antibody from mixed infections with HIV-1 and HIV-2 by serological absorption test.

The interpretation of dual seroreactivity with human immunodeficiency virus type 1 (HIV-1) and HIV-2 in blood samples is a serious problem facing AIDS researchers worldwide. Some samples of sera from HIV-1-infected patients showed a serological cross-reaction with HIV-2, causing confusion regarding the serodiagnosis. Therefore, we tried to differentiate these serum samples from those containing real mixed infections with both types of virus. Sera from patients with HIV-1 infections with HIV-2 cross-reacting antibody in Japan were distinguished from sera from patients with mixed infections with HIV-1 and HIV-2 in West Africa by our serological cross-absorption test, which proved to be highly specific and useful for serodiagnosis.     

A comparison of 2 T-lymphoid cell lines persistently infected with the human immunodeficiency virus (HIV-1) with different productive capacities]

T-lymphoid cell lines (H9/CBL-4 and CEM/CBL-4) persistently infected with HIV-1 were observed simultaneously for 6.5 months. The virus activity was characterized by such parameters as the number of infected cells determined by fluorescent antibody technique, the total level of virus--specific protein synthesis determined by immune blotting method, and the capacity to infect H9 and CEM cells. A comparative analysis of the two cell lines helped define the evaluation criteria for high and low productivity cultures. It was shown that a short-term virus persistence could exist in high-productivity cultures and long-term persistence in low-productivity cultures. The cytopathic activity of virus in cultures could be judged by accumulation of virus protein p24 in cell-free supernatants, this being one of the factors defining the efficacy of infection of H9 and CEM T-lymphoid cells.