CROI 2018 Abstract eBook

Abstract eBook

Oral Abstracts

infectious virion along with 2 copies of dimeric viral genomic RNA and host tRNALys3, which acts as a primer for initiation of reverse transcription. Upon viral entry, initiation is slow and non-processive compared to elongation. We have applied cryo-electron microscopy (cryo-EM) to determine the three- dimensional structure of the HIV RT initiation complex. RT is in an inactive polymerase conformation with open fingers and thumb and the nucleic acid primer-template complex is shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV RNA lies in the cleft of RT, extended by additional pairing interactions. The 5’ end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action. 100 STRUCTURE AND MECHANISM OF THE SAMHD1 HIV-1 RESTRICTION FACTOR Ian A. Taylor , The Francis Crick Institute, London, UK SAMHD1 is a post-entry cellular restriction factor that inhibits HIV-1 replication in myeloid-lineage and resting CD4+ T cells. The mechanism of SAMHD1 restriction has been disputed but the predominant theory is that SAMHD1 dNTP triphosphohydrolase activity blocks HIV-1 infection by reducing the cellular dNTP pool to a level that does not support viral reverse transcription. A large body of structural and biochemical studies have demonstrated that the active form of SAMHD1 is a protein tetramer that contains four regulatory allosteric sites each accommodating a deoxynucleotide/nucleotide pair and four active sites that hydrolyse the dNTP substrates. In addition, other studies have shown that the dNTP triphosphohydrolysis reaction is regulated by tetramer stability, controlled by SAMHD1 phosphorylation at residue T592. However, although, this wealth of information has contributed significantly to our understanding of SAMHD1 restriction, regulation and activation the molecular details of the catalytic mechanism of dNTP hydrolysis have remained unclear. Therefore, to elucidate the molecular mechanism of dNTP triphospho-hydrolysis by SAMHD1, we now have undertaken comprehensive enzymological studies employing deoxynucleotide substrate and activator analogues and determined crystal structures of catalytically active SAMHD1 with dNTP-mimicking, competitive inhibitors. These analogue studies uncovered inhibitors of SAMHD1 and also revealed the capacity for SAMHD1 to be activated by and hydrolyse existing antiviral and anticancer drugs. The SAMHD1-inhibitor co-crystal structures show in atomic detail how dNTP substrates are coordinated at the SAMHD1 active site and reveal how the protein chemically activates a water molecule to mount a nucleophilic attack on the phospho-ester bond in the dNTP substrate. In conclusion, these studies now provide the molecular details of the SAMHD1 reaction mechanism demonstrating how dNTP substrates are hydrolysed and enable more accurate prediction of whether new and existing antiviral and anticancer drugs are hydrolysed by SAMHD1. 101 STRUCTURE AND MECHANISM OF VPS4, THE ENZYME THAT DRIVES HIV BUDDING Christopher P. Hill , University of Utah, Salt Lake City, UT, USA Many cellular membrane fission reactions are driven by ESCRT pathways, which culminate in remodeling and disassembly of ESCRT-III polymers by the AAA ATPase Vps4. HIV-1 and many other viruses recruit an ESRCT pathway in order to bud from cells. Recent advances in understanding of the budding machinery will be summarized, with special emphasis on HIV and findings from our 3.2 Å resolution cryo-EM structure of the active Vps4 hexamer in complex with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Five Vps4 subunits form a helix, with interfaces between the first four of these subunits apparently bound to ADP.BeFx (ATP) and the interface between the fourth and fifth subunit bound to ADP, as if it is just commencing dissociation from the helix. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. The ESCRT-III peptide binds in an extended (beta- strand) conformation against the five helical subunits. Two classes of side chain binding pockets are formed primarily by Vps4 pore loop 1 residues, with four copies of each pocket propagating along the highly solvated pore through the Vps4 hexamer. The pockets accommodate a wide range of residues, while main chain hydrogen bonds help dictate substrate-binding orientation. The structure

supports a ‘conveyor belt’ model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate disengagement at the lagging end. In this manner Vps4 may disassemble ESCRT-III to reveal a metastable membrane configuration that resolves by fission and virus budding. This model likely applies to other ESCRT pathways and may be generally applicable to multiple other protein-translocating AAA ATPases. Caroline Sabin , University College London, London, UK The last decade has seen a dramatic improvement in the life expectancy (LE) of people with HIV (PWH) in settings with access to effective antiretroviral therapy. As a result, there has been a change in the spectrum of clinical events that are now commonly seen in PWH, with a reduction in the incidence of most AIDS events but an increase in the incidence of co-morbidities usually seen in an ageing population. The jury is still out on whether successfully treated PWH experience an increased risk of these age-related co-morbidities compared to their HIV-negative counterparts. This presentation will summarise the latest data on LE and the co-morbidities that are seen in PWH and will consider whether LE has now ‘normalised’ in comparison with that of the HIV-negative population, drawing attention to the possible methodological biases that may be present when undertaking such a comparison. The presentation will also investigate whether there are important subsets of PWH in whom LE remains substantially shorter than desired, and where interventions to further improve LE may be required. In settings where tuberculosis is not the dominant cause of morbidity and mortality among people with HIV (PWH), evidence continues to build showing a greater-than-expected burden of physical and mental comorbidities in PWH, including hypertension, diabetes, renal impairment, and depression. This presentation will highlight the role of traditional, modifiable risk factors, such as smoking, substance use and other untreated mental health conditions, weight changes, and social cohesion, as well as the interplay between physical and mental comorbidities, on the clinical outcomes. The timing of ART initiation and the changing side-effects of ARTs may create sub-groups with different burdens of physical and mental comorbidities. Effective interventions to reduce modifiable risk factors will be highlighted. Future research is needed to determine how to appropriately leverage existing HIV care infrastructures to be vehicles for interventions that reduce the burden of physical and mental comorbidities so that the quality, not just the quantity, of life can be maximized for PWH. Jason V. Baker , Hennepin County Medical Center, Minneapolis, MN, USA Ongoing systemic inflammation contributes to increased clinical risk among contemporary HIV+ patients despite antiretroviral therapy (ART) treatment with viral suppression. This presentation will review epidemiologic data showing that blood biomarkers, reflecting generalized inflammation, innate immune response (e.g., monocyte activation), adaptive immune response (e.g., T-cell activation), and coagulation activity, predict risk for a broad spectrum of end-organ diseases among HIV+ patients. Strategies to mitigate HIV-associated inflammation will then be reviewed. Effective ART with viral suppression reduces inflammation, but the degree of improvement appears incomplete when compared to uninfected persons. In addition, delaying ART treatment (e.g., from delayed diagnosis) may lead to excess inflammation even after viral suppression is achieved. Novel, broadly anti-inflammatory, treatment(s) have shown promise in the general population, but the safety (i.e., risk from infection) among HIV+ individuals requires further study. Ultimately, future research should focus on minimizing residual inflammation with ART treatment, as well as identifying safe anti-inflammatory treatments to be given in addition to ART, for HIV+ patients that remain at excess inflammation-associated disease risk.

102 LIFE EXPECTANCY IN THE MODERN ART ERA

Oral Abstracts

103 IMPACT OF PHYSICAL AND MENTAL COMORBIDITIES ON LIFE EXPECTANCY Keri N. Althoff , Johns Hopkins University, Baltimore, MD, USA

104 INFLAMMATION AND DISEASE RISK AMONG HIV-SEROPOSITIVE INDIVIDUALS

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CROI 2018