CROI 2017 Abstract e-Book

Abstract eBook

Oral Abstracts

for HIV and syphilis and PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae in urine samples, oral and anal swabs. The primary study endpoint was the time to a first bacterial STI: gonorrhoea, chlamydia infection or syphilis. We compared the two study arms according to the intention-to-treat principle. We used time-to-event methods, including Kaplan–Meier survival curves and Cox proportional-hazards models. Results: From July 2015 to January 2016, 232 pts were randomized, 116 in each arm. Median follow-up was 8.7 months (IQR: 7.8-9.7). Seventy-three pts acquired STIs during the study period, 28 pts in the PEP arm (24%, 37.7 events per 100 pt-years) as compared to 45 pts in the no PEP arm (38.8%, 69.7 events per 100 pt-years) for a hazard ratio (HR) of 0.53 (95% CI: 0.33-0.85, P=0.008). HR for gonorrhoea, chlamydia infection and syphilis were 0.83 (95% CI: 0.47-1.47, p=0.52), 0.30 (95% CI: 0.13-0.70, p=0.006) and 0.27 (95% CI: 0.07-0.98, p<0.05), respectively. Overall 71% of all STIs were asymptomatic. Pts in the PEP arm used a median of 7 pills/month (IQR: 3-13). Safety was good with only 8 pts (7%) discontinuing PEP because of gastro-intestinal adverse events (AEs). Gastrointestinal AEs were reported in 61 pts (53%) and 47 pts (41%) in the PEP and no PEP arms, respectively (p=0.07). There was no significant change in sexual behavior between study arms during follow-up. Conclusion: On demand PEP with doxycycline reduced the incidence of chlamydia infection and syphilis in high risk MSM and has an acceptable safety profile. The long-term efficacy of this strategy and its impact on antibiotic resistance needs to be assessed. 92 THE FATES AND FOLDS OF HIV-1 RNA Alice Telesnitsky, Univ of Michigan, Ann Arbor, MI, USA Interconverting RNA structures composed of overlapping sequences near HIV-1 RNA’s 5’ end orchestrate several replication functions. For example, when folded into the packaging signal Ψ, this region of RNA dimerizes and forms a structure that is recognized by viral proteins to mediate specific encapsidation of HIV-1 genomic RNA. The same region of RNA can adopt an alternate fold that is not recognized for packaging and likely contributes to other replication functions, such as serving as mRNA. The importance of these alternate RNA structures is supported by experimental work in which mutations that stabilize the mRNA conformer decrease RNA packaging. However how HIV-1 naturally achieves the correct balance of RNA structures has remained unclear. In work comparing the folding of the packaging signal as a minimal RNA element vs in its native context, we made the surprising discovery that very subtle transcript variation-even the addition of a single nucleotide far from the sequences that fold into the packaging signal-could completely shift the equilibrium of alternate RNA structures. Our analysis suggested a model in which this tiny change could initiate a domino effect of alternating basepairs, and made us speculate that similar transcript heterogeneity could help define the balance of HIV-1 RNA structures if it existed in vivo and had gone undetected in previous analyses. Our subsequent analysis of virion and infected cell RNAs revealed that indeed, the HIV-1 RNAs in infected cells include transcripts that differ subtly from one another, and that virion and infected cell RNA populations differ significantly. Further analyses revealed that this heterogeneity results from integrated HIV-1 proviruses making use of a small cluster of transcription start sites, with the shorter of the resulting transcripts adopting the packaging-competent fold while RNAs longer by a single base or two are enriched on poly-ribosomes, and preferentially adopt the translation-competent fold. These studies represent a striking example of a previously-unrecognized form of RNA regulation, in which the folding of initial transcribed sequences can set up a cascade of RNA folding events that dictate an RNA’s folded conformation, binding partners and biologic destiny. 93 STRUCTURE AND FUNCTION OF IMMATURE AND MATURE HIV CAPSIDS Hans-Georg Kräusslich, Heidelberg Univ, Heidelberg, Germany Formation of infectious HIV particles is directed by the viral Gag polyprotein and occurs at the plasma membrane of the producer cell. Gag initially assembles into an immature capsid shell structured as a truncated sphere consisting of regular hexameric subunits and irregular defects. This stable capsid structure corresponds to the production mode, while Gag proteolysis by the viral protease inside the complete virion converts the structure into the infection mode. Release of the CA domain from the polyprotein leads to disassembly of the immature shell and formation of the cone-shaped mature capsid, exhibiting a fullerene-geometry consisting of CA hexamers and pentamers. Recent cryo electron microscopy analyses have yielded unprecedented details of the structures of both the immature and the mature virion, and provided new insights into capsid function and inhibition. 94 A ROLE OF HIV-1 INTEGRASE IN ENCAPSIDATION OF THE VIRAL RNA GENOME Mamuka Kvaratskhelia, The Ohio State Univ, Columbus, OH, USA An essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosomes is well established. However, mutagenesis studies have suggested a multifunctional role of IN in HIV-1 biology as two distinct phenotypes have been observed for IN substitutions: class I, which selectively impair integration and class II, which display additional reverse transcription and particle maturation defects. However, for the past 20 years it has remained enigmatic as to how IN could contribute to proper viral particle maturation. The interest in elucidating a potential active role of IN during virion maturation has been bolstered recently after the discovery of allosteric HIV-1 IN inhibitors (ALLINIs), which are currently in clinical trials. ALLINIs induce aberrant IN multimerization in virions and similar to class II IN substitutions yield eccentric, non- infectious virions, where ribonucleoprotein complexes are mislocalized outside of the protective capsid core. To elucidate a potential role of IN during virion maturation we used crosslinking-immunoprecipitation sequencing (CLIP-seq) and complementary in vitro biophysical approaches to test whether IN interacts with the viral RNA genome and whether these interactions contribute to correct viral particle morphogenesis. Our studies have led us to the following novel findings: i) IN binds the viral RNA genome in virions. These interactions have specificity as IN exhibits distinct preference for select viral RNA structural elements in virions and in vitro; ii) CLIP-seq results show divergent but highly reproducible binding patterns for IN and nucleocapsid (NC) on the viral RNA genome suggesting that these proteins can bind viral RNA at the same time in the virion; iii) IN R269A/K273A substitutions compromise IN binding to the viral RNA genome without significantly affecting other known functions of this viral protein and result in particles with eccentrically mislocalized ribonucleoprotein complexes; iv) Likewise, ALLINIs impair IN binding to the viral RNA genome in virions of wild type but not the escape mutant virus; v) Unlike IN-RNA interactions, NC-RNA complexes are not affected in the eccentric non-infectious virions. Collectively, these results reveal a non-catalytic biological function of IN during virion maturation, which includes its ability to bind and localize the viral RNA genome within the protective capsid core, and elucidate the mode of action of ALLINIs. 95 TRIM5ALPHA RECOGNITION OF THE HIV CAPSID Barbie K. Ganser-Pornillos, Univ of Virginia, Charlottesville, VA, USA Restriction factors and pattern recognition receptors make up intrinsic cellular defenses against viral infection. TRIM5 proteins are restriction factors and receptors that recognize the incoming capsid cores of retroviruses. Upon capsid binding, TRIM5 proteins accelerate dissociation of the viral core, inhibit reverse transcription of the viral genome, and activate ubiquitin-dependent interferon production. TRIM5 proteins contain the tripartite motif fold (RING, B-box, and coiled-coil domains) and a C-terminal domain (SPRY or CypA) that mediates direct binding to retroviral capsids. Retroviral capsids are fullerene structures composed of about 1,500 copies of the viral CA protein, which are organized on a hexagonal lattice composed of about 350 hexamers and exactly 12 pentamers. Capsid recognition is avidity-driven, requiring higher-order assembly of multiple TRIM5 protein molecules on the capsid surface. We have been using a combination of techniques, including X-ray crystallography of purified TRIM proteins and domains, biochemical reconstitution of TRIM5/capsid complexes, and cryoEM analyses of these complexes to obtain molecular insights on how the TRIM5a protein specifically recognizes the HIV-1 capsid. Our aggregate data (together with those of others) reveal that TRIM5α assembles into a two-dimensional hexagonal net that wraps around a retroviral capsid. The hexagonal TRIM net displays an array of SPRY domains to match the positions and orientations of their corresponding binding epitopes on the capsid surface. Assembly of the TRIM lattice also promotes dimerization and activation of the RING domains, leading to efficient synthesis of polyubiquitin chains. Higher-order assembly of TRIM5α therefore spatially organizes the biochemical activities of the different TRIM5α domains and directly couples capsid recognition to ubiquitin-dependent downstream processes that lead to viral inhibition and interferon signaling.

Oral Abstracts

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CROI 2017