CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

384 Minor Contribution of Host-HIV Readthrough Transcripts to the Level of HIV-1 gag RNA Alexander Pasternak 1 ; Una O’Doherty 2 ; Ben Berkhout 1 1 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands; 2 University of Pennsylvania, Philadelphia, PA, US

Background: Cell-associated (CA) HIV-1 unspliced RNA is an important marker of the viral reservoir and the response to antiretroviral therapy (ART). Recently it has been used in clinical trials as a measure of virus activation by latency-reversing agents. Primers specific for the HIV gag regions are frequently used in PCR-based assays that quantify unspliced RNA. However, because HIV-1 integrates within actively transcribed host genes, it has been suggested that some of the transcripts detected by the gag -specific assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. To properly interpret the results of the gag assays, it is necessary to determine the relative contribution of such readthrough transcripts to the HIV gag RNA in ART-treated patients. Methods: We developed a sensitive nested real-time PCR assay that amplifies the 5’ LTR-encoded U3 – packaging signal region (U3-Psi) of HIV-1. This assay specifically measures host-HIV readthrough transcripts but does not detect genuine HIV-1 unspliced RNA (Fig. 1). Total DNA and total RNA were isolated from PBMC samples of 48 ART-treated patients whose plasma viremia had been undetectable (<40 copies/ml) for ≥ 1 year prior to the study. CA HIV-1 DNA and RNA were separately quantified in these samples using both the U3-Psi assay and the seminested real-time PCR assay specific for the HIV-1 gag region. The sensitivity of both assays is 4 copies/reaction. The same inputs of DNA or RNA were used for both assays. Results: As expected, both U3-Psi and gag assays detected HIV-1 DNA in >90% of the patients (44/48 and 46/48, respectively) with no significant quantitative bias between the assays, demonstrating the functionality of the U3-Psi assay. HIV-1 gag RNA was detected in 44/48 of these patients (92%) with the median copy number of 590 (interquartile range, 217-1194) copies/ m g total RNA. However, the detectability of readthrough RNA was only 40% (19/48 patients). In the 19 patients where the readthrough RNA was detected, its copy number was 49 (41-122) copies/ m g total RNA, representing only 8.3% (2.4%-11.2%) of the HIV-1 gag RNA. Notably, the real readthrough/ gag RNA ratio is much lower, as patients with undetectable readthrough RNA (60% of all patients) were excluded from this calculation.

Figure 1. Schematic representation of real-time PCR assays for the detection of readthrough and gag RNA. LTR, long terminal repeat; ORF, open reading frame; Ψ , HIV packaging signal (Psi). Conclusions: We observed only a minor contribution of host-HIV-1 readthrough transcripts to the level of HIV-1 gag RNA. The vast majority of HIV-1 gag RNA transcripts in ART- treated patients represent genuine HIV-1 unspliced RNA. 379 Characterizing the Active HIV Reservoir on ART: Cell-Associated HIV RNA and Viremia Feiyu Hong ; Elizabeth Fyne; Anthony R. Cillo; Margaret A. Bedison; Dianna Koontz; JohnW. Mellors University of Pittsburgh, Pittsburgh, PA, US Background: Despite combination antiretroviral therapy (ART), HIV-1 RNA can be detected in plasma and peripheral blood mononuclear cells (PBMCs), indicating proviral transcription and production of virions, i.e. an active reservoir of HIV. It is not known whether proviral copy number, HIV-1 transcription, and residual plasma viremia on ART are related. Methods: We conducted a cross-sectional study of viremic patients off ART and of virologically suppressed (<50 cps/ml) patients on ART. PBMCs were tested for total cell- associated (CA) HIV-1 DNA and unspliced HIV-1 RNA using sensitive qPCR targeting 3’ pol. Plasma was tested for residual viremia by single copy assay targeting the same pol region. Unpaired t-test was used to compare viremic and patients on ART. Correlations between plasma viremia and cellular nucleic acids were assessed with Pearson’s coefficient. Results: 12 viremic patients and 23 patients on ART were studied. In patients on ART, median CA HIV-1 DNA was 310 copies/10 6 PBMCs (range: 45, 984) and median CA HIV-1 RNA was 59 copies/10 6 PBMCs (range: 1, 454), both were significantly lower than in viremic patients (median 565 [range: 48, 4680], p = 0.033; median 296 [range: 33, 19172], p = 0.030; for CA HIV-1 DNA and RNA, respectively). The 5-fold reduction in CA HIV-1 RNA on ART is small compared with the > 4 log 10 difference in plasma viremia between these two groups (median 0.44 [range: 0.4, 26] vs. 10542 [range: 564, 474211] copies/mL on and off ART, respectively), indicating substantial persistence of HIV-1 transcription despite ART. A strong, positive correlation was detected between cell-associated HIV-1 DNA and unspliced RNA in both viremic (Pearson’s r = 0.974; p < 0.001) and patients on ART (r =0.779; p <0.001). In viremic patients, the levels of plasma HIV-1 RNA also show strong, positive correlations with cell-associated HIV-1 DNA and RNA (Pearson’s r = 0.849 and 0.843, respectively; p < 0.001). By contrast, in patients on ART, residual plasma viremia was not correlated with cell-associated HIV-1 DNA (r = 0.06; p = 0.78) or RNA (r = -0.18; p = 0.39). Conclusions: This is the first study to show i) a strong, positive correlation between the number of HIV-infected cells and the level of cell-associated HIV-1 RNA in patients on ART, and ii) no correlation between cell-associated HIV-1 RNA and the levels of persistent viremia. These findings suggest that most of persistent HIV-1 transcription in patients on ART does not result in viremia. 390 Nascent LTR-Driven Transcription Can Lead to Translation of HIV Proteins in Resting CD4+ T Cells Laura DeMaster 1 ; Alexander Pasternak 2 ; Una O’Doherty 1 1 University of Pennsylvania, Philadelphia, PA, US; 2 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands Background: We have previously described a model of direct infection of resting CD4+ T cells and contrasted it with models of activated CD4+ T cell infection. We found that infected resting CD4 cells express low levels of viral protein without releasing infectious virus, raising the possibility that reservoirs may express HIV proteins in vivo and be visible to the immune system. Unspliced RNA (usRNA) encoding gag was the predominant HIV RNA form detected in infected resting cells. We designed experiments to ask if nascent transcription occurs in resting CD4+ T cells or if the Gag signal detected is due to an artifact such as read-through transcription or incoming virus. Methods: To address the contribution of incoming virus to Gag signal, we first sorted Gag+ and Gag-negative cells from cultures infected in vitro and measured levels of HIV DNA in both populations, similar to an approach we used in vivo. RT-PCR and FACS analysis were used to determine whether other viral proteins (made from spliced RNA forms) were present in cells infected in vitro and in CD4+ T cells from ART patients. In addition, given that tat/rev is present at very low levels in vivo in patients on ART, we asked if tat/rev is required for LTR driven expression.

Oral Abstracts

110

CROI 2015