CROI 2019 Abstract eBook

Abstract eBook

Oral Abstracts

Methods: We fluorescently labeled CA and evaluated replication of the resulting labeled viruses. We generated dually labeled VSV-G pseudotyped particles containing eGFP-tagged CA (CA-eGFP) and mCherry-tagged, Vpr- transincorporated integrase (IN-mCherry). Since CA-eGFP by itself did not allow viral particle release, we co-transfected plasmids coding for CA-eGFP with a WT CA plasmid at a 1:10 ratio during virus production. At discrete time points after infection, we analyzed the cellular localization of both CA-eGFP and IN-mCherry by confocal microscopy in the absence and the presence of inhibitors of the early HIV replication steps. Results: We investigated the cellular distribution and intensity of fluorescent, CA and IN in HeLa P4 cells. CA and IN colocalized in 20-30% of all cytosolic complexes. Importantly, the intracellular distribution and fluorescence intensity of IN-mCherry complexes were unaffected by CA-eGFP labeling. CA-eGFP complexes accumulated in the perinuclear area, but only 10-15% of these also contained IN-mCherry. Using both CA-eGFP and immunocytochemistry, we confirmed the presence of CA in the nucleus, which rarely (<5%) colocalized with IN-mCherry. Under PF74 treatment, the number of nuclear complexes containing labeled IN decreased 15-fold while CA-eGFP decreased 5-fold, consistent with a PF74-mediated nuclear import block. The inhibition of CA-eGFP labeled viruses with PF74 suggests that at least some of the dually labeled particles undergo bona fide uncoating and nuclear import. When using Ral to block integration, we observe a 25% accumulation of fluorescent IN, but not CA-containing complexes in the nucleus. These data question the role of nuclear CA in integration and urge investigation of other nuclear roles of this protein. Conclusion: Directly labeled CA allows single virus imaging of HIV-1 pre- integration steps and provides insights in the cytosolic and nuclear distribution of CA. Therefore, virions carrying labeled IN and CA represent a suitable system to address HIV-1 entry following both the viral PIC and the fate of the associated capsid. 74 DISRUPTION OF HIV-1 LTR SEQUENCE BY A NUCLEOCAPSID MUTATION LEADS TO DTG RESISTANCE Atsuko Hachiya 1 , Karen A. Kirby 2 , Hirotaka Ode 1 , Maritza Puray-Chavez 3 , Mai Kubota 1 , Urara Shigemi 1 , Reiko Okazaki 1 , Masakazu Matsuda 1 , Mayumi Imahashi 1 , Wataru Sugiura 1 , Yoshiyuki Yokomaku 1 , Yasumasa Iwatani 1 , Stefan Sarafianos 2 1 National Hospital Organization Nagoya Medical Center, Nagoya, Japan, 2 Emory University, Atlanta, GA, USA, 3 University of Missouri, Columbia, MO, USA Background: Dolutegravir (DTG), a key component of ART, tightly binds to the catalytic site of integrase (IN) and to the canonical ‘-CA OH ’ dinucleotide sequence of the LTR at the viral DNA (vDNA) ends. Resistance to DTG is poorly understood. Methods: DTG-resistant viruses were selected using in vitro serial passage experiments under DTG pressure. We monitored the viral dynamics of early steps of HIV-1 replication using multiplex immunofluorescent cell-based detection of viral DNA, RNA and protein (MICDDRP) and qPCR. To elucidate the resistance mechanism, we used next-generation sequencing and analyzed the sequence of the LTR termini of HIV-1 that is integrated into host DNA. Results: Through in vitro passage experiments we discovered that a mutation at the zinc-fingers of HIV nucleocapsid (HIV NC ) enhances DTG resistance ~ 4-fold, by itself, or ~7-fold in the presence of an E157Q polymorphism in the IN region (HIV NC/IN ). We demonstrate that in the absence of DTG, both HIV NC and HIV NC/IN replicate more slowly than wild-type HIV-1 (HIV WT ) without reducing integrated vDNA. MICDDRP and qPCR revealed that HIV NC and HIV NC/IN significantly increase the amount of vDNA during reverse transcription and subsequently integrated them into host genome even at 8h post-infection. Analysis of the virus termini sequences after integration revealed that amount of «normal» ‹-CA OH ’ dinucleotide sequences at the LTR ends was significantly affected: whereas ‘-CA OH ’ was present in HIV WT at 99% and 98% of the 5'- and 3'-LTRs, it was found in 97% and 43% for those of HIV NC , and 79% and 46% for those of HIV NC/IN . Notably, the virus termini sequences formed by HIV NC or HIV NC/IN contained more frequent insertions, deletions, and abnormal LTR ends, which are the substrates of IN and part of the DTG binding site. Conclusion: We report an example of a remarkable epistatic drug resistance mechanism, whereby a mutation in the NC viral gene affects the function of 3 viral proteins, (NC, RT, and IN) resulting in resistance to DTG. We propose that NC changes to affect vDNA formation, which in turn affects the selectivity of DTG binding and its exclusion from the active sites of HIV NC and HIV NC /IN . DTG

resistance is further enhanced by an IN polymorphism, thus highlighting an important role of polymorphisms in IN drug resistance and therapies. 75 INTEGRASE (IN) TETRAMERS ARE THE AUTHENTIC TARGETS FOR ALLOSTERIC HIV-1 IN INHIBITORS Pratibha Chowdary Koneru 1 , Nanjie Deng 2 , Ashley Hoyte 1 , Jared Lindenberger 1 , Stephanie Rebensburg 1 , Ashwanth Francis 3 , Dmitry Lyumkis 4 , Alan N. Engelman 5 , Gregory Melikian 3 , Ronald Levy 6 , Mamuka Kvaratskhelia 1 1 University of Colorado Anschutz Medical Campus, Aurora, CO, USA, 2 Pace University, New York, NY, USA, 3 Emory University, Atlanta, GA, USA, 4 Salk Institute for Biological Sciences, La Jolla, CA, USA, 5 Dana–Farber Cancer Institute, Boston, MA, USA, 6 Temple University, Philadelphia, PA, USA Background: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a new, promising class of antiretroviral agents that disrupt the proper viral maturation by inducing hyper-multimerization of IN and consequently inhibiting its binding to the viral RNA genome. Previous biochemical and crystallographic studies have emphasized the importance of IN catalytic core domain and C-terminal domain for ALLINI induced hyper-multimerization of the protein. Here, we have elucidated a crucial role of the N-terminal domain (NTD) for the ALLINI activity. Specifically, we show the importance of NTD mediated tetramerization of IN for the inhibitor induced hyper-multimerization of the protein. Methods: The separation of different oligomeric states (tetramers, dimers and monomers) of WT IN allowed us to delineate striking selectivity of ALLINI for IN tetramers versus the lower order oligomers. In addition, trans- complementation assays, which allowed us to reconstitute IN tetramers using two dimeric IN mutants, further confirmed the selectivity of ALLINIs for IN tetramers. Based on these findings we have created molecular models of ALLINI mediated tetramer-tetramer interactions. Results: Consistent with the experimental results, the docking scores and free energy calculations indicate that tetramers are preferred over dimers for the formation of ALLINI induced IN polymers. Interestingly, our lead pyridine-based ALLINI KF116 exhibited ~10-fold higher activity (EC50~0.7 nM) against a clinically relevant Dolutegravir (DTG) resistant mutant HIV-1NL4-3(IN N155H/K156N/K211R/E212T) virus compared with its wild type counterpart. Complementary in vitro experiments with recombinant WT and mutant INs revealed that WT IN was a mixture of tetramers, dimers and monomers; whereas under identical conditions the DTG resistant IN (N155H/K156N/K211R/ E212T) predominantly formed tetramers. Conclusion: These observations indicate that ALLINI KF116 is highly complementary to DTG and raise possibilities for the synergetic combination of ALLINIs and INSTIs to further increase the genetic barrier to resistance by limiting HIV-1 options for drug resistant substitutions. Taken together, our biochemical findings coupled with virology experiments show that ALLINIs are highly active during virion maturation and suggests that IN tetramers are formed in virions that are selectively targeted by ALLINIs. 76LB TARGETING VIRUS ENV AND CD44 IMPROVES bNAb AVIDITY AND NEUTRALIZATION POTENCY Djin-Ye Oh 1 , Lihong Liu 1 , Jian Yu 1 , Manoj Nair 1 , Manxue Xia 1 , Sho Iketani 1 , Lili Tsai 1 , Yehuda Z. Cohen 2 , Neal Padte 1 , Yaoxing Huang 1 , David Ho 1 1 Aaron Diamond AIDS Research Center, New York, NY, USA, 2 The Rockefeller University, New York, NY, USA Background: Broadly neutralizing antibodies (bnAbs) hold great promise for the prevention and treatment of HIV infection, but this virus has evolved elaborate ways to evade effective neutralizing antibodies. One of these is the evasion of antibody avidity: Low Env density with spike distances surpassing the average “wingspan” of an IgG impedes inter-spike crosslinking and Env structural constraints hamper intra-spike crosslinking; these limitations of bivalent binding may restrain bnAb potency. Here we hypothesized that bnAb neutralizing activity could be increased through a strategy that overcomes the evasion of Ab avidity by using bispecific Abs (BiAbs) targeting both Env and a host molecule known to be present on the viral surface, CD44. Methods: BiAbs were engineered using the CrossMAb Technology. Neutralizing activity was assessed using the Tzm-bl assay for infectious viruses, including the deCamp global panel. Results: We engineered a prototype BiAb that combines the bnAb PGDM1400 (anti-V2 apex) with the anti-CD44 Ab RG7356 and assessed its neutralizing activity against diverse HIV-1 strains, including primary isolates and the deCamp global panel. As expected, RG7356 had no neutralizing activity. PGDM1400/

Oral Abstracts

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CROI 2019