A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

Functional and structural defects in HIV type 1 nef genes derived from pediatric long-term survivors.

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

Association of antibody reactivity to ELDKWA, a glycoprotein 41 neutralization epitope, with disease progression in children perinatally infected with HIV type 1.

The association between antibody reactivity to the neutralizing epitope ELDKWA in the transmembrane glycoprotein gp41 and disease progression was investigated in 29 children perinatally infected with HIV-1. Levels of antibody reactivity to this epitope, measured over time, were associated with absolute CD4+ lymphocyte numbers and disease status, and inversely associated with the levels of acid-dissociated p24 antigen in the plasma. Early virus isolates from 10 of 12 children with no detectable antibody reactivity to this epitope were sequenced. Only three contained sequences that differed from the consensus, indicating that this epitope is well conserved in this population. None of these three children developed antibodies to the autologous sequences, indicating that at least 80% of children with negative antibody reactivity to this epitope were true nonresponders. Together, these results indicate that the ELDKWA determinant could be an important component in the formulation of a vaccine or for immunotherapeutic approaches to HIV-1 infection.

Longitudinal studies of viral sequence, viral phenotype, and immunologic parameters of human immunodeficiency virus type 1 infection in perinatally infected twins with discordant disease courses.

Perinatal human immunodeficiency virus type 1 (HIV-1) infections cause a broad spectrum of clinical disease and are variable in both the age of the patient at onset of serious disease and the progression of the clinical course. Heterozygotic perinatally infected twins with a marked difference in their clinical courses were monitored during the first 2 years of life. Twin B, the second-born twin, developed AIDS by 6 months of age and died at 22 months of age, while twin A remained minimally symptomatic through the first 2 years. Sequential blood specimens were obtained from the twins in order to characterize the immunologic properties of the children and the phenotypes and genotypes of the HIV-1 isolates at various times. Twin A developed neutralizing antibodies and a high-level antibody-mediated cellular cytotoxicity (ADCC) response, while twin B had no neutralizing antibody and a much lower ADCC response. The virus isolates obtained from the two children at various time points proliferated equally well in peripheral blood mononuclear cells, were nonsyncytium inducing, and could not infect established T-cell lines. They differed in their ability to infect primary macrophages. In parallel to the biological studies, the HIV-1 tat and part of the env gene sequences of the longitudinal isolates at four time points were determined. Sequences of virus from both twins at different time points were highly conserved; the viruses evolved at a similar rate until the last analyzed time point, at which there was a dramatic increase in sequence diversity for the sicker child, especially in the tat gene. Our results show that the viruses isolated at different times do not have significant changes in growth properties. The absence or low levels of neutralizing antibodies may correlate with disease progression in the twins.

Clinical and immunological correlates of immune-complex-dissociated HIV-1 p24 antigen in HIV-1-infected children.

It has been reported that HIV-1 p24 antigen (p24 Ag) detection is improved after dissociation of immune complexes using acid treatment (ICD assay). In order to evaluate the clinical significance of p24 Ag detected by the standard assay and by the ICD assay in pediatric patients, we related these measurements to clinical status, level of p24 antibody, and percentage of CD4+ lymphocytes. Fifty-nine plasma specimens from 20 symptomatic HIV-1-infected children, collected prospectively over a 1-year period, were tested for these markers. Plasma was collected at the beginning of zidovudine therapy and approximately 7 and 12 months thereafter. Compared with the standard assay, the ICD assay showed a higher number of samples positive for p24 Ag (78% versus 34%) and an increase in the levels of p24 Ag (median value of 129 versus 24 pg/ml). The anti-p24 antibody level was inversely correlated with the p24 Ag level measured by either assay. Four children negative for p24 Ag by both assays had a stable clinical course. In contrast, 50% of the children negative by the standard assay but positive for ICD p24 Ag and 75% of the children positive by both assays had progression of disease. No patients were positive by the standard assay but negative by the ICD assay. Children whose plasma tested positive by both assays had lower percentages of lymphocytes that were CD4+ by comparison with children who were negative by both assays; children whose plasma tested positive only by the ICD assay formed an intermediate group.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid complementation assay for anti-HIV-1 drug screening and analysis of envelope protein function.

A complementation assay is described that can be used with relative safety to quantitate rapidly inhibitory effects of potential anti-HIV-1 drugs on virtually any stage of the HIV-1 life cycle by measurements of chloramphenicol acetyltransferase (CAT) activity. Of particular interest is that this system is also capable of detecting inhibition of the viral trans-activator Rev, an important potential target for drug intervention. Other applications of the system may include studies to identify domains of the envelope glycoprotein that determine infectivity and tropism or that define epitopes recognized by neutralization antibodies.

Quantitative analysis of human immunodeficiency virus type 1 antibody reactivity by western immunoblots: evaluation of relative antibody levels in seropositive individuals and mothers.

A quantitative analysis of antibody responses to human immunodeficiency virus type 1 (HIV-1) proteins using Western immunoblots and 125I-labeled protein A is reproducible and can be validated. The antibody levels obtained by Western immunoblots were compared with stoichiometric p24 radioimmunoassay over a wide range of antibody (correlation coefficient, .94; P less than .001). Antibody levels to gp160 and gp120 were validated using purified antigens. Analysis of antibody levels from 31 seropositive individuals revealed a statistically significant correlation between antibody levels to p24 and the other viral proteins except gp120. Anti-gag p24 antibody was strongly correlated with antibodies to other env products, specifically gp41 and gp160. Using the validated assay, HIV-1-infected mothers of infants were found to have highly variable levels of antibody to all viral proteins. Mothers of infected infants did not differ significantly from mothers of uninfected infants in antibody pattern or levels to any viral protein including gp120.

Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.

Human retroviral env and gag polypeptides: serologic assays to measure infection.

Humoral antiviral responses to human retrovirus infections identify persistently infected individuals and can be used to characterize virus-host interactions. Antibodies to native viral polypeptides have been reliably measured, although quantitation of env antibodies is difficult due to a lack of purified antigens. To quantitate antibodies to env antigens, bacterially expressed cloned env polypeptides from the transmembrane regions of human T lymphotropic virus types I and III were applied to nitrocellulose filters in an immunodot assay. A combination of the sensitivity of the Western blot procedure and the specificity of peptides from defined viral sequences was used to detect 49/49 HTLV-III/LAV-infected individuals previously defined as seropositive by radioimmunoprecipitation sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these HTLV-III/LAV envelope seropositive people, 22% lacked antibody to p24 in a radioimmunoassay. In contrast, the sensitivity of antibody detection to HTLV-I env antigens and p24 were comparable. Antibodies to HTLV-I and HTLV-III/LAV env transmembrane peptides were not cross-reactive. Levels of antibody to env antigens of both HTLV-I and HTLV-III/LAV persisted without change for at least 26 mo, suggesting that most infections represent stable virus-host interactions. The use of bacterially expressed env peptides offers a rapid serologic approach for distinguishing human retroviral infections and can be used to define immune responses to specific regions of the viral genome.