CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

sequencing using longitudinal plasma samples from the Women’s Interagency HIV Study (WIHS) cohort to study the development of X4 variants. Methods: We identified 23 subtype B HIV-seropositive WIHS participants meeting the following criteria: 1) CD4 T+ cell counts declined from>500 cell// µl to <100 cells/µl; 2) therapy-naïve or on failed therapy with unsuppressed viral load; and 3) plasma samples available biannually during the period of CD4 decline. Multiplexed Primer ID MiSeq sequencing targeted HIV-1 PR, RT, IN and V1/V3 regions on longitudinal specimens. X4 variants were determined by both criteria: 1) Geno2pheno coreceptor algorithmwith a false positive rate below 2%, and 2) variants form a distinct lineage. Results: Seventeen participants (74%) had X4 variants at the last time point when CD4 was <100 cells/µl and 6 (23%) had R5 only viral populations. We sequenced all available longitudinal samples for eight X4 participants and two R5 participants with an average 6.6 biannual time points per participant. Seven of the eight X4 participants (87.5%) did not have X4 variants at baseline but developed X4 viruses as minority variants (mean abundance 21% in the viral population) at a median CD4 count of 538 cells/µL (IQR: 491-655). One participant had X4 variants at baseline with a CD4 count of 759 cells/µL. Three of the eight X4 participants (37.5%) had rapid CD4 decline (>300 cells/µL per year) after emerging of X4 variants and two of six R5 participants (33.3%) had rapid CD4 decline overall (p = 0.57). Phylogenetic analysis showed that X4 variants could emerge frommultiple pre-existing lineages with distinct V3 sequence changes (Fig 1). Sequence changes at the V1/V2 region were associated with sequence changes in the V3 region during the development of X4 variants in most X4 participants. Conclusion: X4 variants can emerge as minor variants early during the course of HIV infection. The co-evolution of the V1/V2 region and V3 region suggests that the compensatory mutations on the V1/V2 regions may be required to develop X4 tropism.

inhibitors, we assembled a library of purine-like small molecules and screened for molecules that competed FASN from an affinity resin. Our lead molecule, Fasnall, is a thiophenopyrimidine-based molecule that potently inhibited FASN enzymatic activity in cell-based assays and in vitro. In both tissue culture and primary peripheral blood mononuclear cell models of HIV replication, Fasnall inhibited virion production (EC50 = 213 nM) with minimal effects on host cell viability. Conclusion: Our primary result - that efficient HIV-1 replication requires FASN activity - complements previous work that demonstrates enveloped viruses such as hepatitis C virus (HCV), Dengue virus, and human cytomegalovirus (CMV) also require de novo fatty acid biosynthesis. In addition to its importance to viral replication, FASN and FASN-dependent increased de novo fatty acid biosynthesis is associated with breast cancer, melanoma, and hepatocellular carcinoma. In adults, most normal tissues obtain FA exogenously from diet, and as a result, most cells have limited de novo FA biosynthesis and express FASN at very low levels. Thus, de novo fatty acid biosynthesis is minimal in normal cells and required by both intrinsic (cancer) and extrinsic cellular perturbations (e.g. HIV). These features make FASN an attractive target for pharmacological intervention. 177 EXPRESSION OF MDM2 IN MACROPHAGES FAVORS HIV-1 INTEGRATION THROUGH INHIBITION OF P53 Yann Breton , Vincent Desrosiers, Michel Ouellet, Michel J. Tremblay Laval University, Quebec City, QC, Canada Background: Macrophages play an important role in the establishment and propagation of HIV-1 infection. Upon exposure to HIV-1, only a small proportion of macrophages are infected whereas most remain uninfected. To shed light on this issue, transcriptomic analyses were performed to compare infected and bystander populations and determine the molecular basis of HIV-1 permissiveness in macrophages. This study revealed MDM2 as a positive regulator of HIV-1 infection in macrophages. MDM2 is an E3 ubiquitin ligase involved in the DNA damage response and regulates the turn-over of various proteins, including p53. Methods: Monocyte-derived macrophages (MDMs) were transfected with target-specific siRNAs and exposed to a competent R5 HIV-1 virus expressing all viral genes and a small GPI-anchored reporter (HSA). In some experiments, MDMs were treated with chemical inhibitors altering MDM2 functions before being infected. Infection was measured by flow cytometry for HSA expression and by qRT-PCR for the number of integrated HIV-1 genomic DNA copies (Alu- HIV-1 PCR). Results: Knockdown of MDM2 induced a 2-fold decrease in the number of infected macrophages as measured by flow cytometry without affecting cell viability or the expression of viral genes. These results were reproduced with a VSV-G-pseudotyped virus. Integration assays showed a significant reduction in the number of integrated HIV genomes following knockdown of MDM2. These results were confirmed using chemical inhibitors of MDM2. As expected, knockdown of MDM2 by siRNA resulted in a significant increase in mRNA and/ or proteins of p53-induced genes, including p21 ( CDKN1A ). The role of p53 and p21 in the susceptibility of MDMs to HIV-1 were further confirmed with specific siRNAs. Conclusion: Altogether, our results indicate that the resistance to HIV-1 integration associated with MDM2 silencing is independent on the mode of entry and requires the activation of p53. Experiments using the chemical inhibitor Nutlin-3 are of particular interest as this inhibitor specifically blocks the interaction between MDM2 and p53 therefore suggesting that the observed resistance to HIV-1 results from the release/activation of p53 and not the absence of MDM2 per se . The expression level of MDM2 and the activation state of p53 are therefore important factors in the establishment of the infection in MDMs. Identification of viral cofactors regulated by MDM2 will bring a new understanding of signaling events controlling HIV-1 replication in macrophages. 178 INTERPLAY BETWEEN FOLATE CYCLE AND HIV-1 INFECTION IN MONOCYTE-DERIVED MACROPHAGES Vincent Desrosiers , Yann Breton, Michel Ouellet, Michel J. Tremblay Laval University, Quebec City, QC, Canada Background: Macrophages play an important role in HIV-1 infection. These cells are suspected to act as viral reservoirs and thus preclude complete elimination of the virus in infected individuals. A transcriptomic analysis was performed to compare gene expression between non-infected, infected and

Poster Abstracts

176 IDENTIFICATION OF FASNALL, A NOVEL FASN INHIBITOR THAT ATTENUATES HIV-1 REPLICATION

Manjusha Kulkarni 1 , Annette Burkhouse 1 , Menakshi Bhat 1 , Yazan Alwarawrah 2 , Phillip Hughes 2 , Jesus Arcos 1 , David Loiselle 2 , Jordi Torrelles 1 , Nicholas Funderburg 1 , Timothy Haystead 2 , Jesse Kwiek 1 1 The Ohio State University, Columbus, OH, USA, 2 Duke University, Durham, NC, USA Background: HIV-1 engages host machinery to produce progeny, and thereby imposes a substantial burden to cellular metabolism. Thus, metabolic disorders, including body fat redistribution, often occur in people living with HIV-1. Many FA and lipid-biosynthesis enzymes use purine cofactors like ATP and NAPDH. Methods: We performed a functional proteomics screen to define how HIV-1 infection regulates purine-binding proteins. We used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify novel small molecule inhibitors. Results: We observed that HIV upregulates fatty acid synthase (FASN) levels and increases de novo fatty acid synthesis. FASN is a 250kDa, multifunctional enzyme that in the presence of NADPH condenses acetyl-CoA and malonyl- CoA into palmitate. Reduction of FASN levels with siRNA inidcates that FASN is required during a late stage of HIV replication. To identify novel FASN

63

CROI 2018