CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

(downregulated) and adjusted p-value <0.1 were classified as significantly differentially expressed. The differential expression profile of some genes was confirmed by qPCR and western blot. Whole cell extracts were obtained from PBMCs of 33 Study 202 (NCT01338883) HIV+ participants [n=17 from the CCR5/CCR2 antagonist cenicriviroc (CVC) arm; n=16 from the efavirenz (EFV) control arm] were used for western blot analysis of selected factors. Statistical analysis was done using the Mann–Whitney U or Wilcoxon signed-rank tests for unpaired or paired data. Results: CCL2 blocking resulted in the differential expression of 1557 and 117 genes at 4 and 20 h, respectively, in uninfected MDMs, and of 79 and 251 genes at 1 and 4 days in HIV-infected MDMs. Among the up modulated genes annotated in categories related to innate immunity, we focused on the restriction factors Mx2 and APOBEC3A (A3A), whose mechanisms of action may account for the CCL2 blocking-mediated postentry inhibition of HIV replication. The upregulation of both genes by CCL2 blocking was confirmed by qPCR, while an increase of protein level was observed only for A3A in both uninfected and HIV-infected MDMs. In Study 202, A3A levels were increased in CVC (p=0.0004) but not EFV arm at week 48 of treatment, and inversely correlated with soluble CD14 levels (n=33; r=-0.4; p=0.048; Figure 1), which were increased in EFV arm (p=0.04) and slightly reduced in CVC arm. Conclusion: Overall, these data suggest that the CCL2/CCR2 axis may represent a new therapeutic target to strengthen host innate immunity and reduce inflammation thus limiting HIV infection. RF-2011-02347224.

Immunoprecipitation to elucidate the proteins binding to CRM-1, and identified then by Mass Spectrometry Results: We obtained 1680 and 993 proteins interacting with different CRM-1 variants in human cells and murine cells, respectively. Among them, Trim21 exhibited higher affinity for both mCRM-1 and mutCRM-1 (5 and 4 times higher, respectively), in comparison to huCRM-1. This result suggests that Trim21 inhibits the mCRM-1. Sept7, demonstrated similar properties, with lower affinity (2.5 times to mCRM-1 and 1.6 times to mutCRM-1). Additionally, we identified 20 proteins with enhanced association to CRM-1 only in murine cells and only in the presence of HIV. Conclusion: We identified 2 proteins that are potential inhibitors for mCRM-1, but their role in HIV replication should be further investigated. Fully understanding of their functions will significantly promote the use of mice models in studying HIV reproduction and pathogenesis, and development of new effective treatments. 201 NEF ANTAGONIZES TIM-MEDIATED INHIBITION OF HIV-1 RELEASE: ROLE OF SERINCs Minghua Li 1 , Abdul A. Waheed 2 , Eric O. Freed 2 , Shan-Lu Liu 1 1 The Ohio State University, Columbus, OH, USA, 2 National Cancer Institute, Frederick, MD, USA Background: The T cell immunoglobulin and mucin domain (TIM) proteins inhibit release of HIV-1 and other enveloped viruses by interacting with cell- and virion-associated phosphatidylserine (PS). Methods: We examined HIV-1 release that is inhibited by TIM proteins in the presence or absence of Nef and/or SERINCs. Results: We show that the Nef proteins of HIV-1 and other lentiviruses antagonize TIM-mediated restriction. TIM-1 more potently inhibits the release of Nef-deficient relative to Nef-expressing HIV-1, and ectopic expression of Nef, or knockdown of TIM proteins, relieves restriction. HIV-1 Nef does not downregulate TIM-1 expression, but promotes its internalization from the plasma membrane. Intriguingly, depletion of SERINC proteins attenuates TIM-mediated restriction of HIV-1 release, especially that of Nef-deficient viruses, indicating that Nef counteracts TIM-1, at leas in part, through SERINCs. Consistent with this model, MLV glycoGag and EIAV S2 proteins also counteract TIM-mediated inhibition of HIV-1 release. Conclusion: Our work reveals a new role for Nef in antagonizing TIM-1, and highlights a complex interplay between Nef and and HIV-1 restriction by TIMs and SERINCs. 202 IMPACT OF NATURAL HIV-1 NEF POLYMORPHISMS ON SERINC5 ANTAGONISM Steven W. Jin 1 , Nirmin Alsahafi 2 , Xiao Mei T. Kuang 1 , Philip Mwimanzi 1 , Heinrich Gottlinger 3 , Zabrina Brumme 1 , Andrés Finzi 2 , Mark Brockman 1 1 Simon Fraser University, Burnaby, BC, Canada, 2 Centre de Research du Centre Hospitalier de l’Université de Montreal, Montreal, QC, Canada, 3 University of Massachusetts, Worcester, MA, USA Background: Nef enhances HIV-1 infectivity by downregulating host restriction factor SERINC5 from the cell surface. Mutations at several highly-conserved Nef residues impair SERINC5 antagonism, but few studies have evaluated the impact of natural sequence variation on this function. Furthermore, no reports have examined this function for Nef alleles isolated from HIV-1 elite controllers (EC) who display low plasma viral load in the absence of therapy. Methods: Nef alleles from 45 EC and 46 chronic progressors (CP) were isolated from plasma viral RNA. SERINC5 expression on the cell surface was assessed by flow cytometry following co-transfection of CEM T cells with Nef clones and SERINC5-iHA; and results were normalized to wild type Nef (SF2 strain). Natural polymorphisms associated with function were identified by statistical analyses of linked Nef genotype-phenotype datasets and confirmed by site-directed mutagenesis. Infectivity was measured by exposing TZM-bl reporter cells to HIV-1 particles (5ng p24); replication capacity was assessed using Jurkat reporter T cells. Results: EC Nef clones displayed lower ability to downregulate SERINC5 (median 80 [IQR 38-95]% activity vs WT) compared to CP clones (96 [IQR 75- 100]%) (p=0.0005). 18 Nef polymorphisms were associated with differential SERINC5 antagonism, of which eight were confirmed by mutagenesis. Among the eight validated Nef polymorphisms, two that displayed the greatest selective impairment of SERINC5 activity were both HLA class I escape

Poster Abstracts

200 TRIM21 –CRM-1 INTERACTION: A NOVAL MECHANISM FOR HIV RESTRICTION IN MURINE CELLS

Roqaya Sabbah-Abudaabes 1 , Moshe Kotler 1 , Richard Sutton 2 , Hila Elinav 1 1 Hebrew University of Jerusalem, Jerusalem, Israel, 2 Yale University, New Haven, CT, USA Background: Mice have been the favored animal model for researches, but unfortunately many obstacles humper HIV replication in mouse. One of these obstacles is over-splicing of viral RNA (vRNA) in murine cells, which leads to a reduction in RNA transportation that is mediated by Chromosome Region Maintenance-1 (CRM-1) protein. CRM-1 is responsible for transmitting the non- spliced, or partially spliced, vRNA molecules, which are essential for producing infectious virus, to the cytoplasm. Our previous study showed that mouse CRM-1(mCRM-1) is distinct from the human protein (hCRM-1) in 21 amino acids. Three of these amino acids (411, 412, 414) are critical for the export of viral mRNA to the cytoplasm. Interestingly, these amino acids are not the binding site for the viral Rev protein that is the adaptor of the viral RNA to CRM-1. Thus, we hypothesized that another protein binds to this site (411-414), resulting in mCRM-1 inhibition, or hCRM-1 activation.Our goal was to identify proteins that bind differently to CRM1-vRNA complex in mouse and human cells, and to reveal the functional distinctions between the two CRM-1 variants Methods: Gene encoding hCRM-1, mCRM-1 and mutant (mutCRM-1)-which is identical to hCRM-1 except for amino acids in 411, 412 and 414 positions- were cloned into HA-Tag plasmid. We transfected these plasmids to human and mouse cell lines (HEK 293T and B78CyT1 respectively) and co-transfected the plasmids with HIV lentivector, in both cell lines as well. We used Co-

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CROI 2018