CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

173 ERAP2 ADMINISTRATION REDUCES IN VITRO PBMC SUSCEPTIBILITY TO HIV-1-INFECTION Irma Saulle 1 , Salomè Valentina Ibba 1 , Cecilia Vittori 1 , Claudio Fenizia 1 , Federica Piancone 2 , Davide Minisci 3 , Elisa Maria Lori 1 , Daria Trabattoni 1 , Mario Clerici 1 , Mara Biasin 1 1 University of Milan, Milan, Italy, 2 IRCCS Don Gnocchi, Milan, Italy, 3 Luigi Sacco University Hospital, Milan, Italy Background: Haplotype-specific alternative splicing of the endoplasmic reticulum (ER) aminopeptidase type 2 (ERAP2) gene results in either full-length (FL, haplotype A) or alternatively spliced (AS, haplotype B) mRNA. HapA/HapA homozygous (homoA) subjects show a reduced susceptibility to HIV-1 infection, probably secondary to the modulation of antigen processing/presenting machinery. As recently it was reported that ERAP2 can be secreted from plasma membrane in response to activation, we investigated if, once released, ERAP2 still retains its antiviral function Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 30 healthy controls (15 homoA and 15 homoB) were in vitro HIV-infected with or without adding different doses of recombinant human protein ERAP2-FL (rhERAP2-FL) and p24 viral antigen quantification was used to assess viral replication. Seven- days post in vitro HIV-1-infection the percentage of perforin and granzyme- producing CD8+ T Lymphocytes and HLA-ABC-expressing cells were analyzed as well; these two parameters were shown to correlate with endogenous ERAP2 activity. Results: As previously shown homoA subjects were less susceptible to in vitro HIV-1 infection (p< 0,01). Addition of rhERAP2-FL to in vitro HIV-infected cells did not affect cell viability and resulted in a reduction of viral replication in both homoA and homoB individuals with a peak effect observed using 100 ng/ml of the protein (p<0.01 in both cases). This protective effect was independent from an increase of HLA-ABC expression and/or of perforin and granzyme expression by CD8+ lymphocytes Conclusion: The role and the targets of ERAP2-FL in the extracellular milieu are still undisclosed and need further investigation. However, data herein suggest that once added to cell culture ERAP2-FL preserves its protective function against HIV-1 infection, even in homoB subjects who do not genetically produce it. Presumably this defensive feature is mediated through an unconventional mechanism, distinct from immune systemmodulation. 174 INCREASED SAMHD1 CORRELATES WITH ISGS IN HIV-1–INFECTED PATIENTS Maura Statzu , Letizia Santinelli, Claudia Pinacchio, Giancarlo Ceccarelli, Ivano Mezzaroma, Ombretta Turriziani, Vincenzo Vullo, Guido Antonelli, Gabriella d’Ettorre, Carolina Scagnolari Sapienza University of Rome, Rome, Italy Background: SAMHD1 is an inducible host innate immunity restriction factor that inhibits HIV-1 replication. The underlying mechanisms of SAMHD1 transcriptional regulation remains elusive and considerable controversy exists over whether type I IFN can support SAMHD1 production. In order to gain new insights into the role played by SAMHD1 in regulating the natural course of HIV-1 infection, we evaluated SAMHD1 expression and its relationship with the IFN response in vivo. Methods: Peripheral blood mononuclear cells (PBMC) from 335 HIV-1-infected patients, both therapy naïve (n=92) and virological suppressed long-term HAART-treated (n=243), and from 100 gender and age-matched healthy individuals were examined. Demographical and clinical characteristics of patients are reported in Table 1. CD4+ T cells, CD14+monocytes and gut biopsies were also analysed in a subgroup of HIV-1-infected patients on suppressive antiretroviral therapy. Gene expression levels of SAMDH1 and ISGs (MxB, HERC5, IRF7) were evaluated by real-time RT-PCR assays. Results: SAMHD1 levels in HIV-1-positive patients were significantly increased compared to those in healthy donors (p=0.04). Virologically suppressed treated patients exhibited higher SAMHD1 levels than healthy donors (p=0.0008), and naïve patients (p<0.0001). SAMHD1 levels were higher in CD4+ T cells than in CD14+monocytes paired samples (treated patients: p=0.038; healthy donors: p<0.0001). By comparing SAMHD1 expression in CD4+ T cells and CD14+monocytes between HIV-1 infected patients and healthy donors, an increased SAMHD1 expression in these cell subsets was recorded in treated HIV-1 positive patients (patients vs healthy donors, CD4+ T cells: p<0.0001; CD14+monocytes: p<0.0001). We selected a subgroup of 7 out of treated HIV- 1-positive patients with relatively low PBMC SAMHD1 mRNA expression in order

cell line was generated by CRISPR/Cas9. Susceptibility to HIV-1 infection was examined by flow cytometry after infection with a VSV-pseudotyped NL4-3 GFP-expressing virus. Gene expression was assessed by quantitative PCR. Protein expression and phosphorylation were analyzed by immunoblotting. Whole transciptome was assessed by RNA-seq. Results: Vpx-induced degradation of SAMHD1 significantly increased HIV-1 infection in primary macrophages. However, no significant change in infection was seen when SAMHD1 expression was inhibited by either siRNA in macrophages or in the CRISPR/Cas9 knockout cell line model. To assay the role of Vpx, whole transcriptome profiling of macrophages untreated or Vpx transduced was performed. 41 genes were differentially expressed: 14 downregulated and 27 significantly upregulated after Vpx-induced SAMHD1 degradation. Interestingly, 14 out of 27 upregulated genes (52%) were IFN-stimulated genes (ISG), including IFNB1, IRF7 and CXCL10, suggesting a relationship between Vpx and activation of the innate immune system. Identified ISG expression was confirmed and extended in additional donor cell samples. Further evaluation of the pathway underlying innate immune activation after Vpx treatment in macrophages showed enhanced expression of the RNA sensors RIG-I and MDA5 without involvement of DNA sensors. On the contrary, when SAMHD1 expression is downregulated by siRNA or by CRISPR/ Cas9, increased expression of DNA sensors cGAS and STING was found, without any significant effect on RNA sensors. Conclusion: Vpx-mediated degradation enhances innate immune activation that is distinct and independent of SAMHD1 expression. These differences may help explain variability in the pathogenicity and immune control of HIV infections. 172 EVOLUTION-GUIDED STUDIES TO UNDERSTAND THE ANTIVIRAL MECHANISM OF IFITM3 Kazi Rahman , Alex A. Compton National Cancer Institute, Frederick, MD, USA Background: The interferon-induced transmembrane (IFITM) proteins are a group of antiviral factors that inhibit the replication of diverse viruses, including HIV-1, at two stages: restriction of incoming viruses in target cells and inhibition of virion infectivity in producer cells. Evidence points to inhibition of virus-cell fusion as the basis for both antiviral functions, but the precise molecular mechanism is unknown. Recent studies suggest that IFITM genes belong to a family of transmembrane proteins known as Dispanins, which are characterized by two transmembrane domains separated by a conserved intracellular loop (CIL). Whereas IFITM proteins inhibit viral and host membrane fusion, another member of the Dispanin family known as PRRT2 inhibits synaptic vesicle fusion in neurons. Therefore, a comparative evolutionary and biochemical analysis between IFITM proteins and other Dispanin members, such as PRRT2, will uncover the mechanistic basis behind membrane fusion regulation. Methods: Multiple sequence alignments of Dispanin family members were performed to identify regions of conservation and divergence. Residues in IFITM3 that are analogous to functionally important sites in PRRT2 were mutated and tested for impact on antiviral functions. 293T cell lines stably expressing IFITM3 variants were generated and challenged with Influenza A and retroviral pseudotypes to study the inhibition of virus entry, while 293T cells co-transfected with IFITM3 variants and retroviral plasmids were used to study the inhibition of virion infectivity. Results: A single residue change in the CIL of IFITM3, never before studied in the context of its function, resulted in a substantial loss of antiviral activity. Importantly, the analogous residue in PRRT2 is critical for its regulation of synaptic vesicle fusion. Western blot and immunofluorescence analysis indicate that the single mutation disrupts protein function without affecting protein expression or turnover. Conclusion: The identification of a single amino acid residue critical to the function of IFITM3 provides an important tool in the search for the molecular mechanism driving antiviral function. The finding that analogous mutations in IFITM3 in PRRT2 disrupt their respective functions suggests that both proteins similarly remodel host membranes and/or operate via the same downstream effectors. We are currently addressing whether the two proteins contain overlapping interaction partners which coordinate vesicular trafficking and fusion.

Poster Abstracts


CROI 2019

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