Antibody to HIV-1 Tat protein inhibits the replication of virus in culture

Summary The HIV-1 transactivator protein Tat is essential for viral replication. Tat is released from infected cells and can be taken up and transactivate HIV-LTR in LTR-CAT transfected cell lines. The present study shows that the addition of monoclonal antibody to Tat in IIIB and MN-infected cultures reduces the HIV antigen production in a concentration dependent manner. These data suggest that external Tat might be important in the replication of HIV, exerting the effect in a paracrine fashion. Using 1 µg/ml of anti-Tat antibody resulted in a decline of HIV antigen production to 33% and 45% of controls in IIIB and MN infected H9 cells, respectively. A time course experiment showed progressively increased inhibition of replication during 7 days of exposure to anti-Tat antibody, which could be due to increasing Tat concentration. The inhibitory effect of anti-Tat antibodies on the replication of HIV could play an important regulatory role during infection in vivo.     

High yield of parasites and prolonged in vitro culture of Histomonas meleagridis.

Dwyer's medium is a frequently employed culture medium for Histomonas meleagridis with rice powder as an essential ingredient. The effect of adding larger quantities of rice powder to the culture medium and the influence of the size of the rice particles on the growth of H. meleagridis was examined. Increasing the amount of rice powder from the standard amount of 10 to 12 mg to 50 to 100 mg per 12.5 ml medium resulted in approximately a 10-fold increase of parasites. Larger quantities of rice powder did not give better yields. The particle size of the rice powder proved relatively unimportant, although the addition of only large rice powder particles (>250 microm) resulted in a somewhat lesser yield. H. meleagridis cultures could be prolonged from approximately 4 days to at least 2 weeks without subculturing by supplementing the culture medium with rice powder only.     

Development of a sensitive ELISA for HIV-1 p24 antigen using a fluorogenic substrate for monitoring HIV-1 replication in vitro.

A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.     

Virion-associated HIV-1 Vpr: variable amount in virus particles derived from cells upon virus infection or proviral DNA transfection

Human immunodeficiency virus type-1 (HIV-1) Vpr is a virion-associated protein implicated to have a role in AIDS pathogenesis. In regard to the amount of Vpr incorporated into virus particles, the published data vary widely. To address this, we quantitated Vpr in virus particles derived from diverse sources that are used to evaluate the biological effect of Vpr. Virus particles from infected cells showed only a small amount of Vpr. Interestingly, virus particles from cells cotransfected with HIV-1 proviral DNA lacking Vpr coding sequences (NLDeltaVpr) and a Vpr expression plasmid showed a drastic increase (29.4-fold) in the incorporation of Vpr. Furthermore, cotransfection involving NLDeltaVpr and different concentrations of Vpr expression plasmid resulted in virus particles containing Vpr in proportion to the Vpr expression plasmid used. The differences in virus particles with respect to Vpr as revealed by these studies should be taken into account in assessing the effect of Vpr.     

Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization

Background  

HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells.     

Using HIV-1 sequence variability to explore virus biology.

Human immunodeficiency virus type 1 (HIV-1) only recently established an epidemic world-wide infection in the human population. The virus persists in the human host through active replication and is able to avoid clearance by the immune system. Active replication is an important component of the rapid evolutionary potential of HIV-1, a potential which manifests itself in the evolution of immune escape variants, drug resistant variants, and variants with the ability to use different cell surface coreceptors in conjunction with CD4. Multiple zoonotic introductions, compartmentalization of virus replication in the body, and genetic bottlenecks associated with sampling during transmission, antiretroviral therapy, and geographic and/or host population isolation further contribute to the range of sequences present in extant viruses. The sum of the history of all of these phenomena is reflected in HIV-1 sequence variability, and most of these phenomena are ongoing today. Here we review the use of HIV-1 sequence variability to explore its underlying biology.     

Recent insights into HIV-1 Vif

The lentiviruses, including HIV-1 (but excluding equine infectious anemia virus), encode a viral infectivity factor (Vif) protein. Circumstantial evidence suggested that Vif acts to neutralize an inhibitory host defense mechanism, but progress in the field was limited because the identity of the cellular target was unknown. The recent identification of the elusive host cell factor let loose a flood of advances. These findings have revealed a novel innate defense mechanism against retroviruses. In infected cells, the cellular cytidine deaminase APOBEC3G, a relative of the activation-induced deaminase (AID), is encapsidated into assembling virions. The enzyme lies in the virion, waiting to wreak havoc on the viral genome in the next round of virus replication--unless it is first caught by Vif.     

Interleukin-15 enhances HIV-1-driven polyclonal B-cell response in vitro.

Interleukin-15 (IL-15) is a recently described cytokine, produced by monocytes/macrophages, with biological activities similar to IL-2. Since IL-15 was shown to stimulate human B-cell proliferation and immunoglobulin secretion, we investigated its effect on human B-cells stimulated with heat-inactivated human immunodeficiency virus type 1 (iHIV-1) in vitro. We observed a dose-dependent elevation of [3H]-thymidine incorporation and immunoglobulin production by B-cells incubated in the presence of iHIV-1. Moreover, IL-15 stimulated HIV-1-driven B-cell proliferation similarly to IL-2. As to immunoglobulin secretion, IL-15 was able to potentiate the stimulatory effect of IL-10. The highest amounts of iHIV caused a decrease in B-cell proliferation and immunoglobulin secretion to baseline levels, even in the presence of cytokines. These findings indicate that during the late stages of AIDS, when monocytes/macrophages become the major site of viral production, IL-15, in concert with other monocyte-derived cytokines, may promote polyclonal B-cell activation and hypergammaglobulinaemia, which are frequently associated with HIV infection.     

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.     

Transfusion-transmitted virus infection in HIV-1-seropositive patients

Objectives   To investigate the prevalence, persistence and genome heterogeneity of transfusion-transmitted (TTV) in HIV-1-infected patients, a group at high risk both of contracting blood-borne viruses and having viral persistence relating to immunodepression.
Methods   Plasma samples from 238 HIV-1 seropositive subjects and 226 healthy blood donors were examined for TTV-DNA both by polymerase chain reaction (PCR) using primers from the conserved regions in the N22 clone and PCR using primers deduced from the untranslated region (UTR). Direct DNA sequencing and phylogenetic analysis were used to characterize 27 TTV isolates from HIV-1 patients or healthy controls.
Results   Using PCR with the UTR primers, TTV DNA was detected in a very high percentage (> 80%) of samples both from HIV-1 seropositive subjects and from blood donors. Using PCR with N22 primers, shown to detect viral strains associated with hepatitis of unknown etiology, TTV DNA was found in 103 of 238 (43.3%) HIV-1-infected patients and in 22 of 226 (9.7%) blood donors. There was no difference in the prevalence of the TTV DNA in HIV seropositive subjects with regard to clinical features related to immunosuppression, markers of HCV infection or intravenous drug use; presence of TTV DNA was associated significantly only with male gender ( P = 0.003). Persistent or intermittent viremia was detected in plasma samples taken up over a period of 19 months in all (15 of 15) HIV-infected patients tested.
Conclusions   The persistence and high frequency of infection detected by PCR with N22 primers in HIV-1 seropositive patients suggest that further clinical investigation of immunocompromised hosts will provide information to clarify the pathogenic role of TTV.     

Titration of human immunodeficiency virus type 1 (HIV-1) and quantitative analysis of virus expression in vitro using liquid RNA-RNA hybridization

An assay is described for titration of human immunodeficiency virus type 1 (HIV-1) and for quantitative analysis of virus expression in vitro. The assay utilizes a liquid RNA-RNA hybridization method coupled with reversible target capture (RTC) on oligo(dT) derivatized magnetic particles. The assay provides a rapid, specific, and sensitive method for quantitation of HIV-1 RNA molecules present either in cells or in viral particles from cell-free culture media. Chronically infected monocytoid U1.1 cells were found to carry 52 pg HIV-1 RNA per 200,000 cells (160 HIV-1 RNA molecules per cell). In contrast, acutely infected CEM and H9 cells carried 3010 and 4370 pg HIV-1 RNA per 200,000 cells (9040 and 13,110 HIV-1 RNA molecules per cell, respectively). No hybridization was observed with uninfected cells or cells infected with HIV-2, HTLV-I, HTLV-II, or EBV. Use of liquid HIV-1 RNA hybridization in association with HIV-1 protein detection methods permits more complete characterization of HIV-1 expression in host cells than either method alone, and also provides a method for standardizing preparations of virus.     

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.     

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.     

Replication of HIV-1 in vivo and in vitro

A complex relationship exists between HIV and its cellular targets. The lethal effect of HIV on circulating CD4⁺ helper T lymphocytes parallels the degree of the infected individual's immunodeficiency and ultimately the transition to AIDS and death. However, as with other members of the Lentivirus family of retroviruses, the ubiquitous, mobile macrophage is also a prime target for HIV infection, and apparently, in most instances, is the initial infected cell, since most people are infected with a CCR5 chemokine-tropic virus. Unlike the lymphocyte, the macrophage is apparently a more stable viral host, capable of a long infected life as an HIV reservoir and a chronic source of infectious virus. Published in vitro studies have indicated that whereas lymphocytes replicate HIV solely on their plasma membrane, macrophages have been envisaged to predominantly replicate HIV within cytoplasmic vacuoles, and thus have been likened to a “Trojan horse,” when it comes to the immune system. Recent studies have revealed an ingenious way by which the cultured monocyte-derived macrophage (MDM) replicates HIV and releases it into the medium. The key macrophage organelle appears to be what is alternatively referred to as the “late endosome” (LE) or the “multivesicular body” (MVB), which have a short and a long history, respectively. Proof of the association is that chemically, LE/MVB and their vesicles possess several pathopneumonic membrane markers (e.g., CD63) that are found on released HIV particles. The hypothesis is that HIV usurps this vesicle-forming mechanism and employs it for its own replication. Release of the intravacuolar virus from the cell is hypothesized to occur by a process referred to as exocytosis, resulting from the fusion of virus-laden LE/MVB with the plasma membrane of the macrophage. Interestingly, LE/MVB are also involved in the infection stage of MDM by HIV. Close review of the literature reveals that along with the Golgi, which contributes to the formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the “Trojan horse” concept.     

In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection.

We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection.