Relationship between human immunodeficiency virus type 1 (HIV-1)-specific memory cytotoxic T lymphocytes and virus load after recent HIV-1 seroconversion.

Human immunodeficiency virus type 1 (HIV-1)-specific memory, or precursor, cytotoxic T lymphocytes (CTL) in 14 subjects who had recently experienced seroconversion were evaluated with respect to virus set point, defined as plasma HIV-1 RNA level 6 months after seroconversion. Env-, Gag-, Pol-, and Nef-specific precursor CTL were detected in (51)Cr-release assays, using antigen-stimulated peripheral blood mononuclear cells as effectors and B cell lines infected with HIV-1-vaccinia recombinants as targets. All subjects tested had precursor CTL specific to at least 2 HIV-1 antigens. Detection of Env-specific precursor CTL was associated with a high set point (P=.0221). The number of antigens recognized tended to be greater in subjects with higher set points (rho=.45621; P=.1171). Gag-specific precursor CTL frequency correlated inversely with set point (rho=-.8478; P=.0003). Two heterozygotes for a 32-bp deletion in CCR5 had the lowest set points (P=.0220) and highest Gag precursor CTL frequencies (P=.0128). These data suggest that host factors that restrict viral replication may be important determinants of the level of HIV-1-specific precursor CTL.     

Antibody response to the viral negative factor (nef) in HIV-1 infection: a correlate of levels of HIV-1 expression

Antibody responses against the nef gene product of HIV-1 were determined in sequential sera from a longitudinally studied cohort of 194 initially asymptomatic HIV-1-seropositive individuals and 72 individuals who seroconverted for antibodies to HIV-1 structural proteins (gag/env). In the majority of men, nef-specific antibodies, once detected, persisted (67.6%). In some men, nef-specific antibodies were only transiently (6.8%), or intermittently (5.3%), detectable. No nef-specific antibodies were found in the remaining men (20.3%). Nef-specific antibodies were elicited early in infection, but rarely (2/72 men) prior to seroconversion for antibodies to HIV-1 structural proteins. An absent, transient, or intermittent nef-specific antibody response was significantly associated with the absence or disappearance of antibodies to HIV-1 core proteins, with (re)appearance and persistence of HIV-1 core antigen and with the presence of low CD4+ cell numbers, i.e. profiles previously shown to be predictive of rapid disease progression. Although more cases of AIDS and AIDS-related disease (21/86 versus 28/180) occurred in the nef-specific antibody-negative group than in the nef-specific antibody-positive group, this difference did not reach significance.     

HIV-1 Nef is preferentially recognized by CD8 T cells in primary HIV-1 infection despite a relatively high degree of genetic diversity

OBJECTIVE: To compare the magnitude, breadth and protein specificity of HIV-1-specific CD8 T-cell responses against the clade B consensus sequence during primary and chronic HIV-1 infection and to analyze the impact of viral diversity on the localization of detected responses. METHODS: HIV-1-specific CD8 T-cell responses against the clade B consensus sequence in individuals with acute (n = 10), early (n = 19) and chronic (n = 10) infection were longitudinally assessed using an interferon-gamma EliSpot assay. RESULTS: CD8 T-cell responses against clade B consensus sequences were preferentially directed against central regions of Nef during primary HIV-1 infection, despite a relatively higher degree of genetic diversity compared with other subsequently targeted regions. In subjects with acute and early infection, Nef-specific CD8 T-cell responses against the consensus Nef sequence represented 94 and 46% of the total magnitude of HIV-1-specific CD8 T-cell responses, respectively. Subjects with untreated chronic infection exhibited broadly diversified CD8 T-cell responses against more conserved viral regions, with only 17% of virus-specific T-cell responses targeting Nef. The initial immunodominance of Nef persisted in individuals with treated acute infection, but shifted rapidly to Gag, Env and Pol in subjects with continuous antigen exposure. CONCLUSION: These data show that despite relatively high sequence variability, viral regions within the clade B consensus sequence of Nef are preferentially recognized during primary HIV-1 infection. Later diversification of responses to other proteins during prolonged antigen exposure provides evidence of the initial preferential immunogenicity of Nef epitopes compared to similarly conserved regions within other viral proteins.     

HIV-1 infection increases the expression of human endogenous retroviruses type K (HERV-K) in vitro

Antibodies to HERV-K antigens have been linked to HIV-1 infection and expression of HERV-K proteins generates T-cell cytotoxic responses in many cancers. HERV-K RNA and protein abundance was measured in HIV-1-infected and control cells. In vitro exposure of HIV-1 laboratory-adapted and primary isolates on U87MG cells increased the expression of HERV-K RNA in a dose-dependent manner. HERV-K RNA and protein burdens were significantly increased in HIV-1-producing H9 cell lines compared to H9 cells. The expression of HERV-K was synergistically increased in HIV-1-infected PBMCs after stimulation with PMA/ionomycin. Furthermore, the expression of HERV-K in PBMCs, and particularly in CD4(+) T cells, was higher in HIV-1 patients compared to control subjects. The expression of HERV-K might be related to HIV-1 pathogenesis and AIDS-associated cancers.     

Homogeneous quasispecies in 16 out of 17 individuals during very early HIV-1 primary infection.

OBJECTIVE: To measure HIV-1 quasispecies diversity in very recently infected male and female plasma donors. METHODS: HIV-1 RNA testing of blood and plasma donations was used to select anti HIV-1 antibody negative, HIV-1 RNA positive plasma samples from 13 males and four females undergoing primary infection. To determine whether these early viral populations were clonal or oligoclonal, heteroduplex mobility assays were performed on multiple independently generated envelope PCR products. Genetically heterogeneous quasispecies where subcloned and their divergent envelope variants sequenced. RESULTS: Because of frequent plasma donations in this population, HIV-1 RNA quasispecies could be studied during very early primary infection. Heteroduplex mobility assays detected the presence of genetically distinct variants in four of the 17 plasma donors. DNA sequence analysis showed that one case was due to a G to A hyper-mutation event and that two cases were caused by the presence of in-frame insertions/deletions resulting in DNA heteroduplex mobility shifts. The early plasma quasispecies of one female contained highly divergent variants differing by up to 6% substitution and multiple insertions/deletions, a level of divergence unlikely to have been generated de novo following transmission. V3 loop sequences analysis indicated the presence of non-syncitium inducing genotypes in 14 out of 17 primary infection cases. CONCLUSION: Plasma viremia is generally genetically homogeneous even during the very early phase of primary infection when viremia is first detected and still rising exponentially. Evidence for the transmission of multiple variants was detected in only one out of four women and none of 13 men undergoing primary infection with subtype B HIV-1.     

Rapidly and slowly replicating human immunodeficiency virus type 1 isolates can be distinguished according to target-cell tropism in T-cell and monocyte cell lines.

Human immunodeficiency virus type 1 (HIV-1) isolates from various patients were divided into two major groups, rapid/high and slow/low, according to their replication properties in vitro. Rapid/high isolates grow well in cell lines and induce the formation of syncytia in peripheral blood mononuclear cells. In contrast, slow/low isolates do not replicate in cell lines and rarely induce syncytia in peripheral blood mononuclear cells. To understand the differences in replicative capacity of these isolates, a panel of indicator cell lines was used. These cell lines were generated for sensitive detection of HIV-1 isolates and show characteristics of T-lymphoid or monocytoid cells. As a result of infection, chloramphenicol acetyltransferase expression is activated. Rapid/high viruses activate chloramphenicol acetyltransferase expression in T-cell and monocytoid indicator cell lines, whereas slow/low isolates activate chloramphenicol acetyltransferase expression only in monocytoid cell lines. The block in infection of T-lymphoid cells by the slow/low isolates appears to occur early in the infection cycle, prior to the production of the virally encoded tat protein. HIV-1 isolates can thus be distinguished according to target-cell tropism. Monocyte-derived cells seem to be a more general target for the various HIV-1 isolates.     

Identification of cellular proteins that bind to the human immunodeficiency virus type 1 trans-activation-responsive TAR element RNA.

Human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat activates the expression of its viral long terminal repeat (LTR) through a target transactivation-responsive element termed TAR. We have constructed cell lines that constitutively express the HIV-1 Tat protein. Analyses of nuclear proteins from these cells and from matched control cells that do not express Tat have identified three proteins that bind to a radiolabeled HIV-1 TAR RNA probe. These polypeptides are 100 kDa, 62 kDa, and 46 kDa in size. Competition experiments using a wild-type TAR RNA sequence, a biologically inactive mutant sequence of TAR, and an unrelated RNA species demonstrated that these proteins show higher binding affinity to wild-type TAR than to the other two non-trans-activatable sequences. We hypothesize that these cellular proteins may mediate a function necessary in Tat-dependent activation of the LTR. The fact that no differences were seen in the binding profiles of nuclear proteins to TAR RNA in Tat-producing and Tat-nonproducing cells suggests that Tat does not directly interact with TAR.     

Knockdown of ERM family member moesin in host cells increases HIV type 1 replication

Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5?cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ?-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.