CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

accommodation of macaque CD4. Surprisingly, mutations had minimal effect on neutralization properties. Conclusion: Identification of this set of mutations may be of use in the development of clinically relevant vaccine design strategies, and additionally may provide insights into mechanisms underlying cross species viral transmission. 181 STRUCTURAL AND DYNAMIC CHARACTERIZATION OF MCD4-BINDING HIV-1 ENVELOPE GLYCOPROTEINS Rachel Kinzelman 1 , Julie M. Overbaugh 2 , Kelly K. Lee 1 , Thaddeus Davenport 1 1 University of Washington, Seattle, WA, USA, 2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA Background: SHIVs are valuable tools that combine SIV and HIV genes in order to replicate within non-human primates. An ideal SHIV would be capable of infecting non-human primates while retaining antigenicity associated with a wild-type HIV. Since HIV-1 envelope glycoprotein (Env) confers cell tropism, it is necessary to utilize an Env that can infect non-human primate CD4+ T cells. Mutations have been found that allow Env to use macaque CD4 (mCD4) for infection. Two of these mutations, A204E and S375W, enable Env isolates to utilize mCD4, but differ in their antigenicity. As Env is a dynamic protein that can shift between closed/unbound and open/CD4-bound states, this suggests that there may be differences in the mechanisms that allow the two mutations to bind mCD4. Using a combination of structural and biophysical approaches, we aim to correlate Env structure with its interaction with mCD4 and neutralizing antibodies (nAbs). Methods: A204E and S375Wmutations were introduced into BG505 SOSIP.664 constructs. Negative stain electron microscopy (ns-EM) was used to characterize the structure of both constructs and will be used to image the trimers bound to antibody Fabs and mCD4. The structural nature of these Envs was also probed using hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to provide insight into dynamic conformational differences between the mutants. HDX-MS will be used to characterize structural changes in response to CD4 binding. Lastly, biolayer interferometry (BLI) has been used to measure the binding affinities to nAbs and profile their antigenicity. Results: There were no obvious dynamic conformational shifts apparent using HDX-MS with both A204E and S375W. However, BLI and ns-EM suggest that A204E samples both an open and closed conformation in contrast to WT and S375W BG505 that maintain a closed conformation. The nAb 17b, which binds the open conformation, binds to A204E trimers but not to wild-type BG505 or S375W. Surprisingly, A204E also binds PGT145, which recognizes the closed structure, suggesting that it does in part adopt a closed conformation. Conclusion: HIV Env variants encoding A204E and S375W differ in their dynamics and their antigenicity. While S375Wmaintains a closed conformation when unbound to CD4, A204E exhibits the ability to switch conformations without being CD4-bound. Thus, the antigenicity profile of A204E is altered compared to the wild type, while S375W is unchanged. Ongoing studies will define structural changes in response to CD4 binding. 182 MUTATIONS IN THE GP41 ECTODOMAIN CAN CONTRIBUTE TO HIV-1 RESISTANCE TO SMFIS Shigeyoshi Harada 1 , Yuta Hikichi 1 , Tetsuo Narumi 2 , Kazuhisa Yoshimura 1 1 National Institute of Infectious Diseases, Tokyo, Japan, 2 Shizuoka University, Hamamatsu Campus, Shizuoka, Japan Background: Small-molecule fusion inhibitors (smFIs) such as IC9564 can inhibit human immunodeficiency virus type 1 (HIV-1) entry into the cells. Recently, we have developed novel IC9564-derived smFIs as a new class of HIV entry inhibitor. In the present study, we investigated HIV-1 variants selected under smFI pressure to get a better understanding of the smFI-virus interaction. Methods: Resistant variants were induced by culture of HIV-1 89.6-infected PM1 cells in the presence of smFIs. We then constructed infectious 89.6 clones carrying mutations selected in the resistant variants. The susceptibility of the infectious clones to smFIs and other class of entry inhibitors was tested by TZM-bl assay. Results: Selection of 89.6 variants under gradually-increased concentrations of IC9564, OKS3-019 and NAT-078 revealed the sequential selection of 4 mutations (H769P (CT), F522V (FP), M26I (SP) and H72Y (C1)), 3 mutations (R838K (CT), R588K (HR1) and V68I (C1)) and 2 mutations (G594R (DSL) and G600E (DSL)), respectively. Studies with engineered smFI-resistant env variants indicate contribution of amino acid changes in the gp41 ectodomain to smFI resistance.

Unexpectedly, these variants were not only highly resistant to smFIs, but also critically dependent on smFIs for its replication. In addition, these resistant mutants exhibited higher sensitivity to BMS-378806, which preferentially recognizes the metastable closed Env conformation. Conclusion: We found viral mutations in the gp41 ectodomain that contribute to the resistance to smFI derivatives. It can be speculated that gp41 modification by these mutations may induce structural rearrangements resulting in formation of the closed conformation, thereby rendering these viruses dependent on smFIs. These results enhance our understanding of Env complex interactions that influence both HIV-1 entry and susceptibility to smFIs. 183 HIV PROVIRAL TRANSCRIPTION RAPIDLY UPREGULATES BCL3, BIRC2, AND BIRC3 TRANSCRIPTION Joseph P. Casazza , Quang N. Nguyen, David R. Ambrozak, Christine Grech, Jianfei Hu, Sam Darko, Amy Ransier, Farida Laboune, Daniel Douek, Richard A. Koup National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Background: ACH-2 cells usually produce low levels of HIV when unstimulated. Viral production increases dramatically with TNFα stimulation. Prior to stimulation with TNFα, surface staining of HIV Env identifies two ACH-2 populations, one which stains for Env and p24, and one which does not. With TNFα stimulation the HIV Env+ population stains more intensely for Env and produces >90% of the virus found in the supernatant; the Env- population slowly becomes Env+ but produces virus at a much lower rate. We have used the ability to separate Env+ and Env- population using flow cytometry to describe the effect of HIV proviral transcription on the ACH-2 transcriptome immediately prior to, and 3, 6, and 9 hours after TNFα stimulation in Env- and Env+ cells. Methods: ACH-2 cells were dual stained with PG9 and VRC07 and bulk sorted before and 3, 6, and 9 hours post-stimulation with 10U TNFα/ml (N=6 replicates). Cells were immediately spun down, lysed with RNAzol and then frozen. Total RNA was extracted, poly-adenylated RNA purified, fragmented and then reversed transcribed using random hexamers. Illumina ready libraries were generated and sequence by paired-end HiSeq 4000 2x75 reads. Results: In the HIV Env- population the frequency of HIV RNA reads increased from 0.05±0.01% prior to stimulation to 0.53±0.15% 9 hour post-stimulation. In the HIV Env+ population the frequency of HIV RNA reads increased from 3.8±0.4% prior to stimulation to 12.9±2.0% 9 hours post stimulation. In both populations, similar increases between pre-stimulation and 3, 6, and 9 hours frequencies of NFKB2, NFKBIA, REL B and TNFAIP3 message were consistent with similar TNFα and NFκB signaling in both populations. The Env+ population showed an 8.4x increase in BCL3 (P=7.5x10 -12 ), a 5.6x increase in BIRC3 (P=1.1x10 -29 ) and 2.4x increase in BIRC2 (P=1.x10 -7 ) messages 3 hours post-stimulation. Except for BIRC2, these changes persisted 9 hours post -stimulation. These changes were not observed in the Env- population. Conclusion: These data suggest that proviral transcription of HIV DNA results in a rapid increase in cellular anti-apoptotic message. Upregulation of these transcripts could stymie the cells innate antiviral responses, increase the longevity of infected cells and increase viral proliferation. Upregulation of BCL3 could also contribute to non-canonical activation of the NFκB pathway thus further increasing viral production. 184 CHARACTERIZATION OF THE EPITRANSCRIPTOMIC LANDSCAPE OF HIV- INFECTED CELLS Sara Cristinelli , Angela Ciuffi Lausanne University Hospital, Lausanne, Switzerland Background: The study of RNA modifications, today known as epitranscriptomics, is of growing interest. The N6-methyladenosine(m 6 A) and 5-methylcytosine (m 5 C) RNA modifications are abundantly present on mRNA molecules, and impact RNA interactions with other proteins or molecules, thereby affecting cellular processes, such as RNA splicing, export, stability and translation. Recently m 6 A marks were found to be present on HIV transcripts and affect viral replication. However, no study has been performed to date to investigate the impact of HIV replication on the transcript methylation level in the infected cell. We used a productive HIV infection model to explore the landscape of m 6 A and m 5 C marks on the transcriptome of HIV-infected cells over a time period of 36 hours and compared themwith mock-treated cells. Methods: The SupT1 T cell line was infected with a high dose of VSV-G pseudotyped HIVeGFP-based vector to ensure ~80% infection efficiency. Cells were collected at 12, 24 and 36h post-infection for mRNA extraction

Poster Abstracts

66

CROI 2019