Search

HIV-1 Tat protein is essential for virus production. RNA-binding proteins that facilitate Tat production may be absent or downregulated in resting CD4+ T-cells, the main reservoir of latent HIV in people with HIV (PWH) on antiretroviral therapy (ART). In this study, we examined the role of Tat RNA-binding proteins on the expression of Tat and control of latent and productive infection. Affinity purification coupled with mass spectrometry analysis was used to detect binding partners of MS2-tagged tat mRNA in a T cell-line model of HIV latency. The effect of knockdown and overexpression of the proteins of interest on Tat transactivation and translation was assessed by luciferase-based reporter assays and infections with a dual color HIV reporter virus. Out of the 243 interactions identified, knockdown of SRP14 (Signal Recognition Particle 14) negatively affected tat mRNA processing and translation as well as Tat-mediated transactivation, which led to an increase in latent infection. On the other hand, knockdown of HMGB3 (High Mobility Group Box 3) resulted in an increase in Tat transactivation and translation as well as an increase in productive infection. Footprinting experiments revealed that SRP14 and HMGB3 proteins bind to TIM-TAM, a conserved RNA sequence-structure in tat mRNA that functions as a Tat IRES modulator of tat mRNA. Overexpression of SRP14 in resting CD4+ T-cells from patients on ART was sufficient to reverse HIV-1 latency and induce virus production. The role of SRP14 and HMGB3 proteins in controlling HIV Tat expression during latency will be further assessed as potential drug targets.

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerization-inhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.

The clinical utility of a flow cytometric assay (FCA) for intracellular HIV p24 antigen was evaluated in a group of HIV-1-infected subjects. A previously described method, p24-FCA (1), was modified to minimize nonspecific staining and to include irrelevant isotype-matched control antibodies. Blood mononuclear cells from 32 HIV-1 seropositive subjects and 14 HIV-1 seronegative controls were examined. In HIV-1 seropositive individuals, the proportion of CD4+ lymphocytes that bound the p24 monoclonal and the isotype control antibodies were not different. The frequency of cells binding p24 antibodies increased with declining CD4 counts and was highest in patients with low CD4+ lymphocyte counts. Although results of p24-FCA do reflect disease progression, the high nonspecific binding of monoclonal antibodies in infected subjects obscures specific p24 binding and precludes its use as an accurate measure of infection within single cells. Cytometry 33:83–88, 1998. © 1998 Wiley-Liss, Inc.

Monocyte--macrophages are important target cells and reservoirs for HIV. The existing methods for the quantification of infectious virus in HIV stocks are not totally satisfactory for use with macrophage cultures. We have developed an infectious focus assay for the direct quantification of virions infectious for human peripheral blood monocyte-derived macrophages adhering to plastic microtitre plates. The combination of an HIV-1 p24-antigen-specific monoclonal antibody and a beta-galactosidase-linked second antibody resulted in a sensitive and very specific assay. With 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as substrate, the assay proved to be as sensitive as p24 antigen quantification in culture supernatants.

HIV-1 persists in peripheral blood monocytes in individuals receiving highly active antiretroviral therapy (HAART) with viral suppression, despite these cells being poorly susceptible to infection in vitro. Because very few monocytes harbor HIV-1 in vivo, we considered whether a subset of monocytes might be more permissive to infection. We show that a minor CD16+ monocyte subset preferentially harbors HIV-1 in infected individuals on HAART when compared with the majority of monocytes (CD14highCD16−). We confirmed this by in vitro experiments showing that CD16+ monocytes were more susceptible to CCR5-using strains of HIV-1, a finding that is associated with higher CCR5 expression on these cells. CD16+ monocytes were also more permissive to infection with a vesicular stomatitis virus G protein-pseudotyped reporter strain of HIV-1 than the majority of monocytes, suggesting that they are better able to support HIV-1 replication after entry. Consistent with this observation, high molecular mass complexes of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) were observed in CD16+ monocytes that were similar to those observed in highly permissive T cells. In contrast, CD14highCD16− monocytes contained low molecular mass active APOBEC3G, suggesting this is a mechanism of resistance to HIV-1 infection in these cells. Collectively, these data show that CD16+ monocytes are preferentially susceptible to HIV-1 entry, more permissive for replication, and constitute a continuing source of viral persistence during HAART.

Objective: To determine the relative contribution of alveolar macrophages, peripheral blood monocytes (PBM) and peripheral blood lymphocytes (PBL) from HIV-infected individuals to HIV-1 viral load. Methods: Alveolar macrophages were obtained by flexible bronchoscopy, and PBM and PBL by venipuncture from HIV-1-infected individuals. Alveolar macrophages and PBM were purified using immunomagnetic bead selection to deplete CD3+ and CD19+ cells from bronchoalveolar lavage and peripheral blood mononuclear cells, respectively. DNA and mRNA were extracted and gag copy number quantified using polymerase chain reaction (PCR) and reverse transcriptase PCR. The titres of infectious cell-associated HIV-1 in cells were determined by the endpoint dilution coculture technique for alveolar macrophages and PBM. Results: Alveolar macrophages and PBM from HIV-1-infected subjects (n = 11) contained equivalent concentrations of HIV-1 DNA and HIV-1 mRNA as determined by PCR and reverse transcriptase PCR, respectively. Antiretroviral therapy was associated with reduced viral DNA concentrations in alveolar macrophages but not in PBM. PBL had a significantly higher level of proviral DNA and mRNA than alveolar macrophages or PBM. Conclusions: Although alveolar macrophages infected in vitro are more permissive for HIV-1 replication than PBM, this difference could not be demonstrated in vivo.

A homosexual man with histologically confirmed Kaposi's sarcoma remained seronegative for HIV-1, HIV-2, and HTLV-1 on conventional tests over a 4-year period. HIV cultures were also negative on thirteen separate occasions. However, serum antibodies to synthetic peptide analogues of the gp41 and nef regions of HIV-1 were consistently detected on an enzyme immunoassay. Tests with the polymerase chain reaction with primers directed to the gag and env regions were negative. The antigens to which the antibodies were produced might have come from a defective HIV mutant, another retrovirus, or a hitherto unknown "agent of Kaposi's sarcoma" with similar antigenic epitopes.

A solid-phase, enzyme-linked immunospecific assay for measurement of different immunoglobulin classes of human rotavirus antibodies is described. The antigen, which was adsorbed directly to polyvinyl microtiter plates, consisted of a clarified cell culture stock of the simian rotavirus SA 11. The assay was sensitive and reproducible and could readily be calibrated to determine concentrations of each class of antibody. The assay was applied to measurements of rotavirus antibodies in serum, colostrum, milk, and fecal samples. It particularly facilitates investigations of the role of immunoglobulin A antibodies in immunity to rotavirus infections.

Serotype-specific monoclonal antibodies were used to select mutants of SA11 rotavirus that were resistant to neutralization. The antigenic characteristics of these mutants were studied with with a panel of monoclonal antibodies. We isolated one type of mutant which showed a dramatic increase (greater than 10-fold) in resistance to neutralization by hyperimmune antiserum, and this together with other data indicates the presence on the rotavirus major outer shell glycoprotein of an immunodominant antigenic site involved in virus neutralization. The mutants were also useful in classifying neutralizing monoclonal antibodies.

During three months after a family outbreak of diarrhoeal disease, rotavirus-specific immunoglobulins of the IgA, IgG and IgM classes were measured by enzyme-linked immunosorbent assay in faecal extracts from the four people involved. Shortly afterwards, sequential extracts were obtained from another infant after a proven rotavirus infection. Rotavirus infection was diagnosed by electron microscopy in three of the patients from whom acute-phase faecal samples were obtained, and all five patients developed a transient specific-antibody response. Antirotaviral IgA, IgM and IgG all reached peak titres between two and four weeks after infection, then dropped back to undetectable levels after two months. If these findings are confirmed in larger numbers of cases, they will provide the basis for simple diagnosis of recent rotavirus infections, without the need of even a single sample of serum.

The polypeptide specificities on monoclonal antibodies previously derived against the SA11 simian, NIC bovine, and Wa human strains of rotavirus were determined by radio-immunoprecipitation of infected cell lysates. All the monoclonal antibodies derived using NIC and Wa were found to be directed against the major component of the inner capsid, while most of the SAll monoclonas were directed against the major outer capsid glycoprotein. When several SAll glycoprotein-specific monoclonal antibodies were used in competitive binding studies, four distinct epitopes, which correlated with the functional activities of the antibodies, were defined. One epitope appeared most critical for virus neutralization, another was involved to a lesser extent, and the remaining two epitopes seemed to have no role. A possible topographical arrangement of these epitopes is suggested.

Monoclonal antibodies were derived against the SA11 simian, NIC bovine, and Wa human rotavirus strains and characterized by enzyme-linked immunosorbent assay, plaque neutralization, and hemagglutination inhibition. Several strain SA11-specific antibodies were found to have neutralizing and hemagglutination-inhibiting capacity.

Antisera to SA11 virus proteins were prepared by immunizing rabbits with individual polypeptides separated by polyacrylamide gel electrophoresis under reducing or nonreducing conditions; the resulting antisera were characterized by four immunological methods. Results of complement fixation tests with double-shelled rotavirus particles and sera raised against reduced or unreduced proteins of the outer shell of the virus suggested the presence of common antigenic determinants in the outer capsid layers of SA11 and the Northern Ireland strain of calf rotavirus. In this test, antisera to outer shell polypeptides gp34 (O2) and gp25 (O4) cross-reacted with calf rotavirions, whereas those to p62 (O1) and p26 (O3) reacted only with the homologous virus. Antisera to the reduced outer shell proteins of the virus did not neutralize viral infectivity, nor did they possess hemagglutination inhibition activity. Evidence suggesting the presence of type-specific antigenic determinant(s) in the major inner protein p42 (I4) of SA11 virus, capable of inducing neutralizing antibody, is presented and discussed. Antisera produced against unreduced gp34 and p26 polypeptides of the virus contained type-specific neutralizing antibodies. Polypeptide gp34 was also capable of inducing hemagglutination inhibiting antibody. All of the antisera to unreduced polypeptides had agglutinating activity against double-shelled particles of homologous and heterologous rotaviruses.

SUMMARY Fc receptor (FcR) and complement receptor (CR) expression on HIV-infectcd monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HlV-lpv 7 days following isolation, and the expression of FcgI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%. FcRIII from 45%, to 9%. and CR3 from 91%. to 67& 14 days following infection. As these receptors are important for macrophage function, their down-modulation muy contribute to the pathogenesis of HIV-related disease.

Background CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively. Results Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. Conclusion Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.