Role of natural killer cells in a cohort of elite suppressors: low frequency of the protective KIR3DS1 allele and limited inhibition of human immunodeficiency virus type 1 replication in vitro.

Natural killer (NK) cells are associated with the innate immune response and are important in many viral infections. Recent studies indicate that NK cells can control human immunodeficiency virus type 1 (HIV-1) replication. We studied the effect of NK cells on HIV-1 replication in a subpopulation of HIV-1-infected individuals termed elite suppressors (ES) or elite controllers. These patients maintain a clinically undetectable viral load without treatment and thus provide a fascinating cohort in which to study the immunological response to HIV-1. Using an autologous system, we analyzed the effects of NK cells and CD8(+) T cells on viral replication in CD4(+) T lymphoblasts. Although we had postulated that NK cells of ES would be highly effective at controlling viral replication, we found that NK cells from some, but not all, ES were capable of inhibiting replication in the presence of interleukin-2, and the inhibition was less robust than that mediated by CD8(+) T cells. Additionally, we examined whether particular alleles of the KIR receptors, specifically KIR3DS1 and KIR3DL1, or allele-ligand combinations correlated with the control of HIV-1 replication by NK cells and whether any specific KIR alleles were overrepresented in ES. Our ES cohort did not differ from the general population with respect to the frequency of individual KIR. However, of the eight ES studied, the four exhibiting the most NK cell-mediated control of viral replication also had the fewest activating KIR and were haplotype A. Thus, the strong NK cell-mediated inhibition of viral replication is not necessary for the immunological control of HIV-1 in all ES.

S-nitrosylation of N-ethylmaleimide sensitive factor mediates surface expression of AMPA receptors.

Postsynaptic AMPA receptor (AMPAR) trafficking mediates some forms of synaptic plasticity that are modulated by NMDA receptor (NMDAR) activation and N-ethylmaleimide sensitive factor (NSF). We report that NSF is physiologically S-nitrosylated by endogenous, neuronally derived nitric oxide (NO). S-nitrosylation of NSF augments its binding to the AMPAR GluR2 subunit. Surface insertion of GluR2 in response to activation of synaptic NMDARs requires endogenous NO, acting selectively upon the binding of NSF to GluR2. Thus, AMPAR recycling elicited by NMDA neurotransmission is mediated by a cascade involving NMDA activation of neuronal NO synthase to form NO, leading to S-nitrosylation of NSF which is thereby activated, enabling it to bind to GluR2 and promote the receptor's surface expression.

Transmission of human immunodeficiency virus type 1 from a patient who developed AIDS to an elite suppressor.

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml. The mechanisms involved in this control of viral replication remain unclear. Prior studies suggested that these patients, as well as long-term nonprogressors, are infected with defective HIV-1 variants. Other reports have shown that the HLA-B*27 and -B*57 alleles are overrepresented in these patients, suggesting that host factors play a role in the control of viral replication. In order to distinguish between these hypotheses, we studied differences in viral isolates and immune responses of an HIV-1 transmission pair. While both patients are HLA-B*57 positive, the transmitter progressed to AIDS, whereas the recipient, who is also HLA-B*27 positive, is an ES. Isolates from both patients were replication competent and contained the T242N escape mutation in Gag, which is known to decrease viral fitness. While the acquisition of compensatory mutations occurred in isolates from the progressor, a superior HIV-specific CD8(+) T-cell response in the ES appears to have prevented viral replication and thus the evolution toward a more fit variant. In addition, CD8(+) T cells in the ES have selected for a rare mutation in an immunodominant HLA-B*27-restricted Gag epitope, which also has a negative impact on fitness. The results strongly suggest that through direct and indirect mechanisms, CD8(+) T cells in some ES control HIV-1 isolates are capable of causing profound immunosuppression.

The multifactorial nature of HIV-1 latency.

HIV-1 can avoid host immune responses and antiretroviral drugs through the latent infection of resting memory CD4(+) T cells. Recently, latent viral genomes have been shown to reside within the introns of active host genes. Therefore, latency is not simply due to an inaccessibility of the integrated proviruses to the transcriptional machinery. Rather, latency might result from insufficient nuclear levels of the crucial activation-dependent host transcription factors required to overcome the transcriptional interference that is an automatic consequence of the nature of HIV-1 integration sites. In addition, resting cells lack sufficient levels of HIV-1 Tat and Tat-associated activation-dependent host factors that are necessary for processive transcription. Defects at consecutive steps of transcriptional initiation and elongation enable HIV-1 to remain hidden within resting CD4(+) T cells.

Intermittent HIV-1 viremia (Blips) and drug resistance in patients receiving HAART.

CONTEXT: Many patients infected with human immunodeficiency virus type 1 (HIV-1) and receiving highly active antiretroviral therapy experience intermittent episodes of detectable viremia ("blips"), which may raise concerns about drug resistance, lead to costly repeat measurements of viral RNA, and sometimes trigger alterations in therapy. OBJECTIVE: To test the hypothesis that blips represent random biological and statistical variation around mean steady-state HIV-1 RNA levels slightly below 50 copies/mL rather than biologically significant elevations in viremia. DESIGN, SETTING, AND PATIENTS: Between June 19, 2003, and February 9, 2004, patients receiving therapy underwent intensive sampling (every 2-3 days) over 3 to 4 months to define the frequency, magnitude, and duration of blips and their association with drug levels and other clinical variables. Blips were defined as HIV-1 RNA measurements greater than or equal to 50 copies/mL preceded and followed by measurements less than 50 copies/mL without a change in treatment. To determine whether blips result from or lead to drug resistance, an ultrasensitive genotyping assay was used to detect drug resistance mutations before, during, and after blips. Patients were 10 HIV-1-infected asymptomatic adults recruited by clinicians and followed up in the Moore Clinic at the Johns Hopkins Hospital. Patients had suppression of viremia to below 50 copies/mL while receiving a stable antiretroviral regimen for 6 months or longer. MAIN OUTCOME MEASURES: At each time point, plasma HIV-1 RNA levels were measured in 2 independent laboratories and drug resistance mutations were analyzed by clonal sequencing. RESULTS: With the intensive sampling, blips were detected in 9 of 10 patients. Statistical analysis was consistent with random assay variation around a mean viral load below 50 copies/mL. Blips were not concordant on independent testing and had a short duration (median, <3 days) and low magnitude (median, 79 copies/mL). Blip frequency was not associated with demographic, clinical, or treatment variables. Blips did not occur in relation to illness, vaccination, or directly measured antiretroviral drug concentrations. Blips were marginally associated (P = .08) with reported episodes of nonadherence. Most importantly, in approximately 1000 independent clones sequenced for both protease and reverse transcriptase, no new resistance mutations were seen before, during, or shortly after blips. CONCLUSION: Most blips in this population appear to represent random biological and statistical variation around mean HIV-1 levels below 50 copies/mL rather than clinically significant elevations in viremia.

Male fertility is compatible with an Arg(840)Cys substitution in the AR in a large Chinese family affected with divergent phenotypes of AR insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is a disorder of male sexual development caused by an absent or dysfunctional AR. Fertile cases with mild AIS and slightly impaired AR activity had been reported in literature, and their external genitalia were documented to be usually normal or subnormal. We reported here an Arg(840)Cys substitution in the AR gene in a large Chinese pedigree affected with AIS. The mutant gene may result in infertility for some affected males with or without hypospadias. However, it was also observed that the mutation did not affect the fertility of the other patients. The gonadotropin levels for one of these patients were within the normal range. Thus, whether normal levels of the gonadotropins are necessary for the preserved fertility of patients affected with this genetic disorder remains to be elucidated.

Histone deacetylase inhibitor Scriptaid reactivates latent HIV-1 promoter by inducing histone modification in in vitro latency cell lines.

Human immunodeficiency virus type 1 (HIV-1) latency remains a major problem for the eradication of viruses in infected individuals undergoing highly active anti-retroviral therapy. By inhibiting HIV-1 gene expression and virus production, histone deacetylase (HDAC) may contribute to the quiescence of HIV-1 within resting CD4+ T cells. A novel HDAC inhibitor, Scriptaid, has been found to have robust activity and lower toxicity compared to trichostatin A (TSA). We therefore investigated Scriptaid for its capability to reverse HIV-1 latency by inducing HIV-1 activation in the Jurkat T cell line containing latent HIV proviruses. We found that Scriptaid can activate HIV-1 gene expression in these latent infected cells by 2-15-fold over background levels, as analyzed by flow cytometry. Chromatin immunoprecipitation (ChIP) assays further revealed that the Scriptaid increased the acetylation level of histones H3 and H4 at the nucleosome 1 site of the HIV-1 long terminal repeat compared to mock treatment. In addition, Scriptaid can synergize with prostratin or tumor necrosis factor-alpha to activate the HIV-1 promoter, with relatively lower toxicity compared to TSA. These studies suggest the potential of Scriptaid in anti-latency therapies.

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation.

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-kappaB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

The role of protective HCP5 and HLA-C associated polymorphisms in the control of HIV-1 replication in a subset of elite suppressors.

Elite suppressors (ES) are untreated HIV-1-infected patients who maintain undetectable viral loads. A recent whole-genome analysis identified two independent polymorphisms associated with low viral loads in untreated HIV-1 infection. We screened 16 ES; none were positive for the protective HLA complex 5 gene polymorphism, and only four were positive for the protective polymorphism associated with the HLA-C gene. These results suggest that some ES control viremia by mechanisms independent of the newly-identified genetic factors.

Orientation-dependent regulation of integrated HIV-1 expression by host gene transcriptional readthrough.

Integrated HIV-1 genomes are found within actively transcribed host genes in latently infected CD4(+) T cells. Readthrough transcription of the host gene might therefore suppress HIV-1 gene expression and promote the latent infection that allows viral persistence in patients on therapy. To address the effect of host gene readthrough, we used homologous recombination to insert HIV-1 genomes in either orientation into an identical position within an intron of an actively transcribed host gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT). Constructs were engineered to permit or block readthrough transcription of HPRT. Readthrough transcription inhibited HIV-1 gene expression for convergently orientated provirus but enhanced HIV-1 gene expression when HIV-1 was in the same orientation as the host gene. Orientation had a >10-fold effect on HIV-1 gene expression. Due to the nature of HIV-1 integration sites in vivo, this orientation-dependent regulation can influence the vast majority of infected cells and adds complexity to the maintenance of latency.

Experimental approaches to the study of HIV-1 latency.

Viral latency is a reversibly non-productive state of infection that allows some viruses to evade host immune responses. As a consequence of its tropism for activated CD4(+) T cells, HIV-1 can establish latent infection in resting memory CD4(+) T cells, which are generated when activated CD4(+) T cells return to a quiescent state. Latent HIV-1 persists as a stably integrated but transcriptionally silent provirus. In this state, the virus is unaffected by immune responses or antiretroviral drugs, and this latent reservoir in resting CD4(+) T cells is a major barrier to curing the infection. Unfortunately, there is no simple assay to measure the number of latently infected cells in a patient, nor is there an entirely representative in vitro model in which to explore the molecular mechanisms of latency. This Review will consider current approaches to the analysis of HIV-1 latency both in vivo and in vitro.

G-->A hypermutation in protease and reverse transcriptase regions of human immunodeficiency virus type 1 residing in resting CD4+ T cells in vivo.

In vitro studies have shown that the host cytidine deaminase APOBEC3G causes lethal hypermutation in human immunodeficiency virus type 1 reverse transcripts unless its incorporation into virions is blocked by Vif. By examining stably archived sequences in resting CD4+ T cells, we show that hypermutation occurs in most if not all infected individuals. Hypermutated sequences comprised >9% of archived species in resting CD4+ T cells but were not found in plasma virus. Mutations occurred in predicted contexts, with notable hotspots. Thus, defects in Vif function in vivo give rise to hypermutated viral genomes that can be integrated but do not produce progeny viruses.

Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes.

Resting CD4+ T-cell populations from human immunodeficiency virus type 1 (HIV-1)-infected individuals include cells with integrated HIV-1 DNA. In individuals showing suppression of viremia during highly active antiretroviral therapy (HAART), resting CD4+ T-cell populations do not produce virus without cellular activation. To determine whether the nonproductive nature of the infection in resting CD4+ T cells is due to retroviral integration into chromosomal regions that are repressive for transcription, we used inverse PCR to characterize the HIV-1 integration sites in vivo in resting CD4+ T cells from patients on HAART. Of 74 integration sites from 16 patients, 93% resided within transcription units, usually within introns. Integration was random with respect to transcriptional orientation relative to the host gene and with respect to position within the host gene. Of integration sites within well-characterized genes, 91% (51 of 56) were in genes that were actively expressed in resting CD4+ T cells, as directly demonstrated by reverse transcriptase PCR (RT-PCR). These results predict that HIV-1 sequences may be included in the primary transcripts of host genes as part of rapidly degraded introns. RT-PCR experiments confirmed the presence of HIV-1 sequences within transcripts initiating upstream of the HIV-1 transcription start site. Taken together, these results demonstrate that HIV-1 genomes reside within actively transcribed host genes in resting CD4+ T cells in vivo.