HIV-1 Nef Targets HDAC6 to Assure Viral Production and Virus Infection.

Sara Marrero-Hernández,Silvia Pérez-Yanes,Jonathan Barroso-González,Romina Cabrera-Rodríguez,Judith Estévez-Herrera,Julià Blanco,Daniel Márquez-Arce,José-David Machado,Agustín Valenzuela-Fernández,Ricardo Madrid

doi:10.3389/fmicb.2019.02437

Abstract

HIV Nef is a central auxiliary protein in HIV infection and pathogenesis. Our results indicate that HDAC6 promotes the aggresome/autophagic degradation of the viral polyprotein Pr55Gag to inhibit HIV-1 production. Nef counteracts this antiviral activity of HDAC6 by inducing its degradation and subsequently stabilizing Pr55Gag and Vif viral proteins. Nef appears to neutralize HDAC6 by an acidic/endosomal-lysosomal processing and does not need the downregulation function, since data obtained with the non-associated cell-surface Nef-G2A mutant – the cytoplasmic location of HDAC6 – together with studies with chemical inhibitors and other Nef mutants, point to this direction. Hence, the polyproline rich region P72xxP75 (69–77 aa) and the di-Leucin motif in the Nef-ExxxLL160-165 sequence of Nef, appear to be responsible for HDAC6 clearance and, therefore, required for this novel Nef proviral function. Nef and Nef-G2A co-immunoprecipitate with HDAC6, whereas the Nef-PPAA mutant showed a reduced interaction with the anti-HIV-1 enzyme. Thus, the P72xxP75 motif appears to be responsible, directly or indirectly, for the interaction of Nef with HDAC6. Remarkably, by neutralizing HDAC6, Nef assures Pr55Gag location and aggregation at plasma membrane, as observed by TIRFM, promotes viral egress, and enhances the infectivity of viral particles. Consequently, our results suggest that HDAC6 acts as an anti-HIV-1 restriction factor, limiting viral production and infection by targeting Pr55Gag and Vif. This function is counteracted by functional HIV-1 Nef, in order to assure viral production and infection capacities. The interplay between HIV-1 Nef and cellular HDAC6 may determine viral infection and pathogenesis, representing both molecules as key targets to battling HIV.

Highlights

Human immunodeficiency virus type 1 (HIV-1) has developed multiple strategies to evade the immune system and to establish a persistent and chronic infection (Moir et al, 2011; Towers and Noursadeghi, 2014; Pyndiah et al, 2015; Sauter and Kirchhoff, 2016; Sumner et al, 2017)
Living HEK-293T cells, transiently over-expressing Pr55GagEGFP, alone or with wt-Nef-DsRed and/or wt-histone deacetylase 6 (HDAC6)-ECFP, seeded in poli-D-Lysine coated coverslips were imaged at 48 h from transfection in chambers containing a KrebsHEPES buffer with 2 mM Ca2+, with an inverted microscope Zeiss 200M (Zeiss, Jena, Germany) through a 1.45 NA objective using an immersion fluid (n488 = 1.518, Zeiss) and under TIRF illumination, as we reported for HIV-1 and cellular studies (BarrosoGonzalez et al, 2009a,b; Garcia-Exposito et al, 2011)
We monitored low levels of p62 protein in MG132 treated cells compared to control, vehicle-treated cells (Figure 2B, MG132 blot), being indicative of a more active autophagic pathway, even under Nef-mediated HDAC6 degradative experimental conditions

Summary

Introduction

Human immunodeficiency virus type 1 (HIV-1) has developed multiple strategies to evade the immune system and to establish a persistent and chronic infection (Moir et al, 2011; Towers and Noursadeghi, 2014; Pyndiah et al, 2015; Sauter and Kirchhoff, 2016; Sumner et al, 2017). Nef fosters a favorable environment for viral replication, subverting a plethora of host cell factors and functions (Arold et al, 1997; O’Neill et al, 2006; Kirchhoff et al, 2008; Lindwasser et al, 2008; Noviello et al, 2008; Foster et al, 2011). Nef promotes either degradation or intracellular sequestration of host receptors, including restriction factors and immune cell receptors, thereby facilitating the escape of HIV-1 from the immune responses (Sugden et al, 2016). Nef captures AP-1 to facilitate the endocytosis and sequestration of major histocompatibility complex type I (MHCI) molecules (Roeth et al, 2004; Jia et al, 2012; Pawlak and Dikeakos, 2015; Dirk et al, 2016), limiting recognition of infected cells by the immune system (Collins et al, 1998). The ability of infected cells to respond to CD28-mediated stimulation would depend on the amount of intracellular Nef, maybe helping in the mechanism of HIV-1 latency (Pawlak et al, 2018)

Objectives

Methods

Results

Conclusion