CROI 2016 Abstract eBook

Abstract Listing

Oral Abstracts

136

SERINC3 and SERINC5 Are Novel Antiviral Proteins Antagonized by HIV-1 Nef Yuanfei Wu ;Yoshiko Usami; Heinrich G. Gottlinger Univ of Massachusetts Med Sch, Worcester, MA, USA

Background: HIV-1 Nef is required for efficient virus replication in vivo . One function of Nef is to enhance the infectivity of progeny virions. Recent studies shown that the glycoGag protein of Moloney murine leukemia virus mimics the effect of Nef on HIV-1 infectivity. This suggests that Nef and glycoGag target a common antiviral factor. Methods: Progeny virions produced by T lymphoid cells were purified in OptiPrep gradients, and virus-associated host proteins were identified by mass spectrometry. The incorporation of tagged SERINC3 and SERINC5 into virions was examined by immunoblotting. The impact of SERINCs on HIV-1 infectivity was examined by siRNA-mediated depletion. T lymphoid cells lacking SERNIC3, SERINC5, or both were generated using the CRISPR-Cas9 knockout approach. The infectivity of progeny virions was examined by measuring beta-galactosidase activity in infected TZM-bl indicator cells, or by infecting primary cells with HIV-GFP. HIV-1 replication was monitored by p24 ELISA. Virus entry was examined with the Blam-Vpr fusion assay. SERINC3 and SERINC5 expression levels were examined by RNAseq and qRT-PCR. Results: Our proteomic analysis of HIV-1 virions indicated that Nef and glycoGag inhibit the incorporation of the multipass transmembrane protein SERINC3 into virions. A biochemical analysis confirmed this finding, and showed that Nef and glycoGag also inhibit the incorporation of SERINC5. The simultaneous knockdown of SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV infectivity. Furthermore, the infectivity of Nef-deficient HIV-1 virions increased more than 100-fold when produced in double-knockout CD4+ T cells lacking SERINC3 and SERINC5, and re-expression of SERINC3 and SERINC5 confirmed that their absence accounted for the infectivity enhancement. SERINC3 and SERINC5 together also restricted HIV-1 replication, and this restriction was counteracted by Nef. Conclusions: We identified the plasma membrane proteins SERINC3 and SERINC5 as novel restriction factors that are incorporated into progeny virions in the absence of Nef and inhibit HIV-1 infectivity. Nef and glycoGag counteract the SERINCs by inducing their endocytosis. Since SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, preventing their downregulation by Nef is a potential anti-HIV strategy. 137 The PPIP122-125 Motif in HIV-1 CA Is an Essential Assembly and Maturation Element Mariia Novikova 1 ; Muthukumar Balasubramaniam 1 ; Sagar Kudchodkar 1 ; Ferri Soheilian 2 ; AnnaT. Gres 3 ; Karen A. Kirby 4 ; Stefan G. Sarafianos 3 ; Eric O. Freed 1 1 NCI, Frederick, MD, USA; 2 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 3 Univ of Missouri, Columbia, MO, USA; 4 Univ of Missouri Sch of Med, Columbia, MO, USA Background: During HIV-1 particle assembly, Gag molecules bind to the plasma membrane and form a hexameric protein lattice, an assembly process driven largely by the capsid (CA) domain of Gag. Upon cleavage of the Gag polyprotein by the viral protease, the released CA proteins form a cone-shaped capsid core within mature virions. In this study, we focused on characterizing the role of a highly conserved PPIP motif (CA residues 122-125) in the loop connecting helices 6 and 7 (H6-7) in assembly, release, and maturation of HIV-1 particles. Methods: We performed alanine-scanning mutagenesis of the PPIP122-125 motif and characterized assembly, production, replication, and infectivity of mutant viral particles. Structural properties of mutant CA proteins and virions were analyzed by EM and x-ray crystallography. Compensatory mutations that rescued the defects imposed by changes in the PPIP motif were also selected through prolonged viral passage in multiple T-cell lines. Results: We show that mutations P122A and I124A significantly decrease production of mature particles and severely inhibit viral infectivity. In addition, both mutants are highly impaired in their ability to replicate in T-cell lines. In contrast, mutation of P123 and P125 residues does not significantly affect viral replication. EM analysis of P122A and I124A mutants revealed abnormal virion structures containing a discontinuous immature Gag lattice. We show that suppressor mutations V11I/T58A are able to rescue the replication defects of both P122A and I124A mutants. Another combination of compensatory mutations, T58S/T107I, was found to rescue the P122A mutant. We demonstrate that the T58A/T107I/P122A mutant is also more sensitive to the CA-based inhibitor PF74. We are currently characterizing the properties of mature viral cores containing P122A or I124A substitutions along with corresponding compensatory mutations. Structural characteristics of the mutant and revertant CA proteins are being investigated by x-ray crystallography. Conclusions: We identified and characterized a novel structural element, the PPIP122-125 motif, which is important for HIV-1 assembly and maturation. Based on the analysis of the Gag lattice structure in the immature virion (Schur et al ., Nature 2015), we suggest that the H6-7 loop of the HIV-1 CA domain plays a role in formation of interhexamer contacts in the immature Gag lattice and also plays an important role in CA-CA contacts in the mature CA lattice.

Oral Abstracts

51

CROI 2016