CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

and FACS analysis. M 6 A RNA modifications were investigated by methylated RNA immunoprecipitation followed by sequencing (MeRIP-Seq). M 5 C RNA modifications were investigated using a bisulfite conversion approach followed by sequencing (BS-Seq). Untouched mRNAs were used as input controls. Libraries were prepared using TruSeq stranded mRNA protocols (Illumina) and sequenced on Illumina HiSeq2500. Results: We obtained a total of 707 million reads. Upon quality control, filtering, and genome alignment we obtained between 8.3 and 40.6 million aligned reads depending on the sample. Preliminary analyses identified transcript methylation as well as multiple genes displaying differential methylation upon HIV infection. Conclusion: Our results highlight the presence of RNA modifications and their potential modulation by HIV, and provide a valuable resource for epitranscriptomic analyses. Therefore, RNA methylation offers a new layer of possible regulation for HIV replication, as well as an array of novel putative therapeutic opportunities to block HIV. 185LB AN HIV E-MAP REVEALS GENETIC INTERACTIONS MEDIATING HIV INFECTION David E. Gordon 1 , Ariane Watson 2 , Assen Roguev 1 , Nevan J. Krogan 1 1 University of California San Francisco, San Francisco, CA, USA, 2 University College Dublin, Dublin, Ireland Background: Functional genetic screens using RNAi and CRISPR-Cas9 are useful for identifying host genes mediating viral infection, however individual genes identified in conventional genetic screens are sometimes difficult to place into the cellular complexes and pathways in which they function. Pairwise genetic interaction screens offer an enhanced approach to studying gene function, permitting for the quantification of functional relationships between genetic perturbations, facilitating the characterization of protein complexes and hypothesis generation regarding gene function. In this proof-of-principle study we have applied genetic interaction mapping to investigate the genes mediating HIV infection in human cells. We present a HIV viral epistasis map (vE-MAP) constructed by pairwise knockdown of 356 human genes in human cells (>63,000 unique combinations). Methods: We generated a combinatorial knockdown matrix of 356 HIV host- dependency factors (>63,000 unique combinations) in cultured human cells and utilized high-throughput microscopy and luminometry to quantify genetic interactions impacting HIV infection. Human genes of interest identified in the vE-MAP screen were studied in primary CD4+ T-cells utilizing Cas9-RNP single and combinatorial knockouts. Results: Hierarchical clustering of vE-MAP data highlights known human protein complexes and resolves structural submodules of the eIF3 complex. In addition to combinatorial knockdown perturbations, we also demonstrate that gene knockdowns may be combined with small molecules and viral mutants to gain insights into their function. In a novel discovery, the vE-MAP identifies numerous negative genetic interactions between the CNOT complex and known HIV host-dependency factors, several of which we validate in primary CD4+ T-cells. Finally, we observe that HIV infection in primary CD4+ T-cells requires CNOT1, 10 and 11 for suppression of type 1 interferon response. Conclusion: This study establishes a foundation for viral genetic interaction mapping utilizing host genetic perturbations, viral mutations and small molecule treatments. We report that the CNOT complex is required for HIV infection in primary T-cells via suppression of the innate immune response.

Poster Abstracts

186 ELUCIDATING THE ROLE OF THE PPIP MOTIF IN HIV-1 CAPSID IN POSTENTRY EVENTS Mariia Novikova 1 , Anna T. Gres 2 , Ryan C. Burdick 1 , Mohamed Husen M. Munshi 1 , KyeongEun Lee 1 , Vineet KewalRamani 1 , Vinay K. Pathak 1 , Stefan Sarafianos 2 , Eric O. Freed 1 1 National Cancer Institute, Frederick, MD, USA, 2 University of Missouri, Columbia, MO, USA Background: The HIV-1 capsid (CA) protein plays multiple roles in the viral replication cycle. As a domain in Gag, CA drives the formation of the immature Gag lattice. Upon maturation, CA reassembles to form the conical core which encompasses the viral RNA genome. During the early stages of the viral replication cycle, CA is involved in a number of processes, including uncoating, recognition by host cellular proteins and nuclear import. Recently, we demonstrated that a highly conserved proline-rich motif (PPIP122-125) in the short loop between CA helices 6 and 7 is an important element for virion assembly. In this study, we characterize the role of the CA PPIP motif in early stages of HIV-1 infection. Methods: We selected for compensatory mutations that rescue assembly and maturation defects of the original PPIP mutants. Replication kinetics, nuclear import efficiency, and host cell restriction factor sensitivity of mutant viruses were analyzed in different cell lines and physiologically relevant cell types. Structures of mutant CA proteins were determined by X-ray crystallography. Nuclear import kinetics were characterized by light microscopy using APOBEC3F-labeled viral complexes. Results: A set of replication-competent viruses including T58S/T107I/P122A, V11I/T58A/P122A, T58A/I124A and V11I/T58A/I124A have been analyzed in this study. Although T58A/I124A and V11I/T58A/I124A mutants are replication competent in PBMCs and parental Jurkat cells, they are highly replication defective in cyclophilin A (CypA)-deficient Jurkat cells and monocyte-derived macrophages (MDMs). We further demonstrated that V11I/T58A/I124A virions enter the nucleus faster than WT virus and are insensitive to cyclosporine A treatment. Upon propagation in CypA-deficient Jurkat cells, the V11I/ T58A/I124A virus acquired a mutation, I124V, which restores its replication competency. Structural analysis by X-ray crystallography revealed that the above-described mutations alter intersubunit interactions and induce subtle changes in the PPIP- and CypA-binding loops. Conclusion: Our findings demonstrate that the PPIP loop in CA modulates the interaction of the incoming viral core with host cell factors, including CypA and potentially nuclear import factors. These results expand our knowledge of post- entry functions of CA and the role of host proteins in productive HIV-1 infection. 187 SINGLE-CELL ANALYSIS REVEALS P2X-DEPENDENT HIV-STIMULATED CALCIUM SIGNALING Tracey L. Freeman , Arpit Dave, Alexandra Soare, Hagerah Malik, Kristin Beaumont, Benjamin K. Chen, Talia Swartz

67

CROI 2019