The emerging role of HLA‐C in HIV‐1 infection

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87109/1/j.1365-2567.2011.03474.x.pd

Effector memory differentiation increases detection of replication-competent HIV-l in resting CD4+ T cells from virally suppressed individuals.

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells

Characterization of LINE-1 Ribonucleoprotein Particles

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1–4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection

LINE-1 ribonucleoprotein particle retrotransposition intermediates.

LINE-1s (L1s) are non-LTR retrotransposons that mobilize (i.e. , retrotranspose) using an RNA intermediate. There are ∼517,000 L1s in the average human genome, ∼80-100 of which are active. Full length active L1s contain a 5' UTR, two open reading frames (ORF1 and ORF2), and a 3' UTR. ORF1 encodes an RNA-binding protein (ORF1p), whereas ORF2 encodes a protein (ORF2p) with endonuclease (EN) and reverse transcriptase (RT) activities. It is hypothesized that only one molecule of ORF2p is synthesized per L1 transcript, whereas ORF1p is produced at greater quantities to coat the L1 RNA. The L1-encoded proteins demonstrate a cis-preference; i.e., they preferentially associate with their encoding transcript to form a ribonucleoprotein particle (RNP), which is hypothesized to be the cytoplasmic retrotransposition intermediate. The L1 RNP forms in the cytoplasm and only after it gains access to the nucleus is the L1 cDNA synthesized by target primed reverse transcription (TPRT). Here, I developed a system to follow the fate of wild type and mutant ORF1p against a background of endogenously expressed L1s in cultured human cells. Using this system, wild type ORF1p and L1 RNA were shown to co-localize in a cytoplasmic RNP, and two classes of mutants in the ORF1p nucleic acid binding domain were discovered that prove RNP formation to be necessary but not sufficient for L1 retrotransposition. I developed a biochemical assay to detect specifically the L1 ORF2p in RNPs via its RT activity, called LEAP, for L&barbelow;ine E&barbelow;lement A&barbelow;mplification P&barbelow;rotocol. Using the LEAP assay, I demonstrated biochemical properties of the L1 RT that are congruent with analysis of genomic integration sites in vivo. Consistent with cis-preference, the LEAP assay also showed the L1 RT prefers its own template and does not promiscuously utilize other cellular RNAs in trans. In sum, these data strongly support the hypothesis that RNPs are bona fide cytoplasmic intermediates, and by the co-localization of ORF2p to the RNP, offers major mechanistic advancements to L1 biology.Ph.D.Biological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125414/2/3192686.pd

Potential Utility of Natural Killer Cells for Eliminating Cells Harboring Reactivated Latent HIV-1 Following the Removal of CD8+ T Cell-Mediated Pro-Latency Effect(s)

An impediment to curing HIV-1 infection is the persistence of latently infected cells in ART-treated people living with HIV (PLWH). A key strategy for curing HIV-1 infection is to activate transcription and translation of latent virus using latency reversing agents (LRAs) and eliminate cells harboring reactivated virus via viral cytopathic effect or immune clearance. In this review, we provide an overview of available LRAs and their use in clinical trials. Furthermore, we describe recent data suggesting that CD8+ T cells promote HIV-1 latency in the context of ART, even in the presence of LRAs, which might at least partially explain the clinical inefficiency of previous “shock and kill” trials. Here, we propose a novel cure strategy called “unlock, shock, disarm, and kill”. The general premise of this strategy is to shut down the pro-latency function(s) of CD8+ T cells, use LRAs to reverse HIV-1 latency, counteract anti-apoptotic molecules, and engage natural killer (NK) cells to mediate the killing of cells harboring reactivated latent HIV-1