CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

163

SPLICING IN A PANEL OF HIV-1 TRANSMITTED/FOUNDER VIRUSES Ann Emery , Ronald Swanstrom Univ of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Background: During HIV-1 replication over 100 spliced RNA variants are generated from a single full-length viral transcript. All spliced transcripts use donor site D1 and one of nine downstream acceptors (A1-A7). About half of the spliced RNAs also undergo further splicing to remove the D4 – A7 env intron (creating the large/4 kb size class that retains the env intron and the small/1.8 kb class of viral RNAs). Additional splicing complexity comes from the use of two additional donor sites, D2 and D3, that create two small exons, one or both of which may be included in splice variants for the downstream genes. The purpose of these small exons is unknown. We examined the extent of splicing pattern variation among 8 transmitted/founder (T/F) isolates to explore the extent of natural variation in splicing patterns. Methods: We have developed a new HIV-1 splicing assay that uses deep sequencing technology with Primer ID-tagged cDNA primers to quantify HIV-1 splicing. In this assay the presence of different spliced variants is quantified within the 4 kb and 1.8 kb size classes. Total infected cell RNA was extracted and used in cDNA reactions using primers specific for each size class, followed by PCR and paired-end deep sequencing using the MiSeq platform. Results: Analysis of 8 subtype B T/F viruses showed that overall splicing patterns were similar in that the same splice site acceptors and donors were used, including conservation of the small exons. However there were examples of surprising ranges of variability including significant changes in the use of alternative splice sites for rev, and over-splicing to the vpr splice acceptor that was compensated by high level use of the adjacent donor D3. We also found evidence of trans-splicing among all of the T/F viruses, where a donor from one transcript splices to an upstream acceptor on another transcript. Conclusion: The idea that splicing is tightly regulated and carefully balanced needs reexamination. The variance in the amounts of the different spliced transcripts suggests that if a virus meets a threshold level for each transcript type, it can be transmitted and continue to replicate. The overall frequency of trans-splicing was surprisingly high, with total trans-splicing occurring more often than several of the canonical spliced transcript types. Trans-splicing was much less frequent in the 4 kb class suggesting this alternative export pathway for viral mRNA experiences a subtly different splicing environment. 164 THE ROLES OF 5 CONSERVED LENTIVIRAL RNA STRUCTURES IN HIV-1 REPLICATION Yang Liu 1 , Olga A. Nikolaitchik 1 , Belete A. Desimmie 1 , Vinay K. Pathak 1 , Kevin M. Weeks 2 , Wei-Shau Hu 1 1 NCI, Frederick, MD, USA, 2 Univ of North Carolina at Chapel Hill, Chapel Hill, NC, USA Background: HIV-1 full-length RNA genome forms complex structures. Some of the RNA structures are known to play important roles in viral replication, which include RNA export, synthesis of GagPol polyprotein, and RNA genome packaging, whereas the functions of other RNA structures are unknown. Using chemical probing, the Weeks’ group has determined the in virio RNA structures of HIV-1, SIVcpz and SIVmac. Comparison of these three RNAs revealed five conserved structures with unknown functions: three (A1 to A3) are located between regions encoding domains of polyproteins, specifically between matrix and capsid (A1), protease and reverse transcriptase (RT) (A2), and RT and integrase (A3). Two of the structures (B1 and B2) form long helical stacks in the region encoding capsid and nef, respectively. We sought to determine whether these RNA structures are important to HIV-1 replication, and if so, their potential functions. Methods: For each RNA structure, we introduced synonymous mutations in NL4-3 to disrupt base-pairing and examined the effects of these mutations in virus production, viral infectivity, and the ability to undergo multiple rounds of viral replication in T cells. We also performed competition experiments to compare the replication fitness of these mutants with that of the wild-type NL4-3 virus. Results: We observed that all five mutants can generate infectious viruses; furthermore, the virus production and one-round replication infectivity of the mutant viruses are not significantly different from those of wild-type NL4-3. We then performed multi-round competition assays between a mutant virus and the wild-type NL4-3 in T cells. We found that three of the mutants have replication fitness similar to that of wild-type virus, suggesting that the loss of these structures do not affect HIV-1 replication in T cells. However, mutations in the A1 or the A3 RNA structures result in loss of replication fitness. We are currently performing additional experiments to determine how these mutations contribute to the loss of replication fitness. Conclusion: We have examined the roles of five conserved RNA structures on HIV-1 replication. Three of the structures are dispensable for HIV-1 replication. However, HIV-1 replication fitness was reduced when synonymous mutations were introduced into sequences between regions encoding matrix and capsid, and RT and integrase; these two RNA structures are likely to be important for efficient HIV-1 replication. 165 EXPONENTIAL GROWTH OF HIV DEPENDENT ON BURST SIZE BREAKTHROUGH OF THE ALLEE THRESHOLD Background: Exponential growth is the mode by which a population establishes itself following arrival in a new environment. For HIV, establishment is inevitable by the time exponential growth is detected, during both acute infection following transmission, and viral rebound from the latent reservoir following interruption of suppressive antiretroviral therapy (ART). Although fundamental, the processes leading up to overt exponential growth versus extinction have eluded definition because they are simultaneously low amplitude, rare, and fleeting. Methods: To investigate viral release and growth, resting CD4+ T cells from 8 donors on ART were stimulated with antibodies against CD3, CD2, and CD28. The cells were cultured in limiting dilution with ART to quantify initial HIV release in the absence of new infections, or with IL-2 plus exogenous cells for outgrowth. To distinguish replication-competency from establishment, supernatant positive for HIV RNA by RT-PCR on day 8 of the primary outgrowth culture was transferred to new stimulated CD4+ cells from HIV-uninfected donors. Fitting the experimental results to deterministic and stochastic models, we tested key assumptions of HIV release and early growth, and present an adapted model that not only fit existing data but was predictive of more complex experiments. Results: Despite extensive cell division, most CD4+ T cells initially present in culture died before virus could be released, which on average began 4 days following activation. The duration of HIV release due to one latent cell ranged from less than one day up to 7 consecutive days. Such sustained detections resulted from sequentially occurring progeny releasing on average 1000 HIV RNA copies per cell for a total 3900 HIV RNA copies. Many primary culture releases consisted of virus which was confirmed intact by secondary culture, but did not result in exponential growth. Released HIV in a well was most likely to undergo exponential growth if it exceeded a critical threshold of 6000 HIV RNA copies. Establishment dependence on a low threshold is called the Allee effect and has been previously reported across diverse biological taxa including animals, plants, and non- pathogenic microorganisms. Conclusion: This work demonstrates why integrated and intact provirus may not result in ex-vivo outgrowth, advances the theoretical foundation for rebound prediction following ART interruption, and identifies the Allee growth threshold as a population dynamically defined target for an HIV functional cure. Jason M. Hataye 1 , Joseph P. Casazza 1 , David R. Ambrozak 1 , Takuya Yamamoto 2 , Daniel Douek 3 , Alan S. Perelson 4 , Richard A. Koup 3 1 NIH, Bethesda, MD, USA, 2 Osaka Univ, Sukta, Osaka, Japan, 3 NIAID, Bethesda, MD, USA, 4 Los Alamos Natl Lab, Los Alamos, NM, USA

Poster and Themed Discussion Abstracts

64

CROI 2017