CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

194 NEF DIMERIZATION REGULATES HIV-1 INFECTIVITY AND SERINC5 INCORPORATION Ryan Staudt , Thomas E. Smithgall University of Pittsburgh, Pittsburgh, PA, USA Background: Nef is an HIV-1 accessory factor essential for viral pathogenesis and immune escape. Nef lacks intrinsic biochemical activity, functioning instead via interactions with multiple host cell effectors related to signaling and endocytic trafficking. Nef function also depends on self-association through conserved hydrophobic residues that form a dimer interface (L112, Y115, F121). Mutation of these residues disrupts diverse Nef functions, including enhancement of viral infectivity, replication, and receptor downregulation. Here we investigated whether Nef dimerization is linked to sensitivity to the host cell restriction factor SERINC5 (S5), a potent suppressor of infectivity that is antagonized by Nef. Methods: Viruses were produced in 293T cells transfected with HIV-1 NL4-3 proviral DNA in the presence or absence of an expression vector for S5 with a C-terminal HA tag. Viral supernatants were harvested 48 h later and assessed for infectivity using TZM-bl reporter cells. S5 incorporation was assessed by immunoblotting of HIV-1 virions following purification by ultracentrifugation. Results: Consistent with previous reports, co-expression of S5 in producer 293T cells severely impaired the infectivity of Nef-defective HIV-1 (∆Nef). The infectivity of wild-type HIV-1 produced in the presence of S5 was higher due to the expression of Nef, which excludes S5 from the cell membrane to prevent incorporation into the envelope of budding virions. Interestingly, the infectivity of virions produced from proviruses with Nef dimerization interface mutations showed reduced infectivity approaching that of the ∆Nef virus, and infectivity was reduced even further upon co-expression of S5. Immunoblotting showed an increase in S5 incorporation into purified virions with Nef dimerization interface mutants similar to that observed with the ∆Nef virus. Conclusion: The infectivity of HIV-1 virions expressing Nef dimer interface mutants is impaired to a similar extent as the Nef-defective virus when produced in the presence of S5, suggesting that Nef homodimer formation is important for S5 antagonism. This conclusion is supported by biochemical data, where S5 incorporation is increased in newly synthesized virions in the presence of these Nef mutations. However, the infectivity of Nef mutant viruses is also impaired in the absence of S5 expression, suggesting that additional mechanisms control infectivity of viruses produced in 293T cells that are S5- independent. 195 LIPID BINDING DOMAIN IMPACT ON HIV-1 NEF AND SRC-FAMILY KINASE HCK U-SH3-SH2 COMPLEX John Alvarado , Haibin Shi, Thomas E. Smithgall University of Pittsburgh, Pittsburgh, PA, USA Background: The HIV-1 accessory protein Nef supports high-titer viral replication, immune evasion of HIV-infected cells, and is essential for AIDS progression. Efficient replication of HIV-1 in primary human macrophages requires expression of the myeloid Src-family kinase, Hck. Nef provides a crucial link between HIV-1 and this host cell kinase, interacting with Hck through its SH3 domain and driving constitutive kinase activation. Both Nef and Hck are myristoylated at their N-termini, resulting in co-localization to the cytoplasmic face of cellular membranes. Also contributing to membrane localization are the anchor and unique domains of Nef and Hck, respectively, which are positioned N-terminal to the Nef core and Hck SH3 domain. To better understand the role of these N-terminal lipid binding domains on Nef homodimerization and Hck recruitment, we are pursuing the X-ray crystal structure of full-length Nef (FL-Nef; SF2 allele) in complex with the Hck unique-SH3-SH2 (Hck-U32) regulatory domains. Methods: We developed an E. coli expression system for the expression and purification of soluble Hck-U32 tandem regulatory domains. Stable interaction between purified Hck-U32 and FL-Nef was then determined using analytical size-exclusion chromatography (SEC) and surface plasmon resonance (SPR). The Hck-U32 and FL-Nef expression systems were combined to co-purify the two proteins as a complex (FL-Nef:Hck-U32) by immobilized metal affinity chromatography and SEC. Crystallization trials of this purified complex are in progress using the sitting-drop vapor diffusion method. Results: Using our expression system, the Hck-U32 protein has been successfully expressed and purified from E. coli in soluble form. Hck-U32 and FL-Nef form stable complexes in solution as demonstrated by both analytical SEC and SPR. The SPR analysis suggests a high affinity interaction between

Hck-U32 and FL-Nef with a kinetic K D value in the low μM range. In addition, the recombinant FL-Nef:Hck-U32 complex has been purified to homogeneity and is currently in crystallization trials. Conclusion: The Hck-U32 protein is amenable to expression and purification in soluble form and stably interacts with FL-Nef in solution, enabling structural analysis of the complex by X-ray crystallography. This complex structure is anticipated to yield fresh insight into the role of these N-terminal lipid binding domains in the regulation of protein:protein interactions at biological membranes. 196 IP-10 PRODUCTION BY THE LYMPH NODES MEDIATES ENTRY OF SIV- SPECIFIC CXCR3+CD8+ T CELLS Antonio Valentin 1 , Eric Ramirez 1 , Hrishikesh Pandit 1 , Ashli Earl 1 , Kate E. Broderick 2 , Niranjan Sardesai 2 , George N. Pavlakis 1 , Barbara K. Felber 1 1 National Cancer Institute, Frederick, MD, USA, 2 Inovio Pharmaceuticals, Inc, Plymouth Meeting, PA, USA Background: Lymph nodes (LN) harbor cells chronically infected by HIV/SIV, especially CD4+ follicular helper lymphocytes which are located within the B cell areas. LN are characterized by low frequency of effector memory CD8 T cells that are, in general, excluded from the follicles. Using a vaccination protocol that combines intramuscular DNA delivery followed by in vivo electroporation, we have analyzed the trafficking of virus-specific CD8+ T lymphocytes. Methods: Eight MamuA01+ rhesus macaques were immunized with plasmid DNA encoding p57gag. Two weeks after the fourth vaccination, the animals were sacrificed, and the dissemination of vaccine-induced T cell responses was monitored throughout the body by immunophenotyping combined with CM9gag tetramer staining, followed by flow cytometry. Chemokine production, including CXCL9, CXCL10 and CXCL11, by lymph node mononuclear cells (LNMC) from the vaccinated animals stimulated ex vivo with SEB and IFNγ was measured by ELISA, multiplex chemiluminescence detection assays (MSD) and intracellular staining. Results: High magnitude (up to 15% of total CD8+ T cells) of vaccine-induced virus-specific CD8+ T cells were found in peripheral blood from all the immunized macaques. These cells were actively dividing (ki67), expressed high levels of CXCR3 and efficiently migrated into central and peripheral LN. The CM9-specific CD8+ T cells within the LN also expressed CXCR3 and had an effector phenotype (CD95+CD28lowCD45RAlowCD127-CCR6-CCR4-) with no significant CCR7 expression, suggesting that these cells could be located outside the T cell areas. LNMC from these vaccinated macaques stimulated ex vivo with IFN-γ or SEB released high levels of IP-10 (CXCL10) and CXCL11. A combination of surface and intracellular staining with anti-CXCL9 and IP-10 antibodies revealed that these chemokines were produced by HLA-DR+ B lymphocytes, CD11c+CD14- dendritic cells and a subset of cells with the phenotype (CD3- CD20-CD14-CD11c-HLA-DR-CD21+) of follicular dendritic cells (FDC). Conclusion: Expression of CXCR3 by the vaccine-induced virus-specific CD8+ T cells indicates that these cells can migrate into areas where the chemokines CXCL9, CXCL10 and CXCL11 are produced. Because these chemokines are produced within the LN by cells located in the B cell areas (B lymphocytes and FDC) the data suggest that a CXCR3-dependent and CXCR5-independent pathway of effector cells entry into the follicles exist. 197 LYMPH NODE TREG SUBSETS ARE EXPANDED IN SOME HIV+ PEOPLE ON SUPPRESSIVE ART Joy M. Folkvord 1 , Lishomwa C. Ndhlovu 2 , Brooks I. Mitchell 2 , Fredrick Yost 2 , Martin McCarter 3 , Amie Meditz 3 , Cecilia M. Shikuma 2 , Elizabeth Connick 1 1 University of Arizona, Tucson, AZ, USA, 2 University of Hawaii at Manoa, Honolulu, HI, USA, 3 University of Colorado Anschutz Medical Campus, Aurora, CO, USA Background: Tregs including follicular regulatory T cells (TFR) are expanded in lymph nodes of untreated HIV+ people. TFR impair TFH responses and humoral immunity in untreated disease. Little is known about the impact of antiretroviral therapy (ART) on Treg and TFR populations in secondary lymphoid organs. Methods: Frequencies of Tregs (FoxP3+CD8-) and TFR (Tregs located in CD20+ B cell follicles) were determined by immunostaining of formalin fixed paraffin embedded lymph node (LN) tissue sections and quantitative image analysis. Frequencies of HIV RNA+ cells were determined in the same tissue section using in situ hybridization (RNAscope). Data were analyzed using descriptive and nonparametric statistics.

Poster Abstracts

70

CROI 2019

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