HIV-1 antisense protein of different clades induces autophagy and associates to the autophagy factor p62

Abstract Over recent years, strong support argues for the existence of an HIV-1 protein encoded by antisense transcripts and termed Antisense Protein (ASP). Furthermore, a recent in silico analysis has provided evidence for its recent appearance in the genome of HIV-1. We have previously detected ASP in various mammalian cell lines by Western blot (WB), flow cytometry and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. The aim of the current study was to examine autophagy induction by testing ASP from different clades, and to identify the implicated autophagy factors. We firstly confirmed that NL4.3-derived ASP was interacting with itself and that multimer formation was dependent on its amino region. Removal of this region was associated with reduced level of induced autophagy, as assessed by autophagosome formation but deletion of the most amino cysteine triplet did not totally abrogate multimer and autophagosome formation. Expression vectors of ASP from different clades were next tested and led to detection of monomers and varying levels of multimers with concomitant induced autophagy, as determined by increased LC3-II and decreased p62 (SQSTM1) levels. Through confocal microscopy, ASP was noted to co-localize with p62 and LC3-II in autophagosome-like cellular structures. CRISPR-based knock-out of ATG5, ATG7 and p62 genes led to increased stability in the levels of ASP. Furthermore, co-immunoprecipitation experiments demonstrated the interaction between p62 and ASP, which was dependent on the PB1 domain of p62. Interestingly, immunoprecipitation experiments further supported that ASP is ubiquitinated and that ubiquitination was also responsible for the modulation of its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy- and Ubiquitin/Proteasome System (UPS)-mediated degradation in which ubiquitin is playing an important role. Understanding the mechanisms underlying the degradation of ASP is essential to better assess its function. Author Summary In the present study, we provide the first evidence that a new HIV-1 protein termed ASP when derived from different clades act similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective material. We have gained further knowledge on the mechanism mediate the activation of autophagy and have identified an important interacting partner. Our studies have important ramification in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated Neurological Disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies are thus important and demonstrate the importance in conducting further studies on this protein, as it could represent a new interesting target for antiretroviral therapies and vaccine design.

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