HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

Abstract

The existence of the antisense transcript-encoded HIV-1 antisense protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blotting, flow cytometry, and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Here, our goals were to assess autophagy induction by ASP from different clades and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino-terminal region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASPs from different clades (A, B, C, D, and G) were tested next and were detected in monomeric and multimeric forms at various levels, and all induced autophagy (clade A ASP was less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knockout of ATG5, ATG7, and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP colocalized with p62 and LC3-II in autophagosome-like structures. Coimmunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported the idea that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function. IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramifications 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 hence demonstrate the importance in conducting further studies on this protein as it could represent a new interesting target for antiretroviral therapies and vaccine design.

The HIV-1 Antisense Protein ASP Is a Transmembrane Protein of the Cell Surface and an Integral Protein of the Viral Envelope

“…However, the use of a single cell type, a single technique, or tagged ASP overexpressed ectopically through transient transfection (in part due to the lack of specific antibodies against ASP) did not permit general conclusions about its expression in the context of HIV-1 infection to be reached. Moreover, the role of ASP in the HIV-1 life cycle remains to be elucidated (26,42).…”

“…Moreover, the demonstration that ASP is an integral protein of the cellular and viral envelope also suggests a possible role during viral budding and/or entry. Two recent studies have shown that ASP from all HIV-1 clades has the ability to induce autophagy (26,42). However, the advantage that triggering this pathway would provide to HIV-1 replication was not investigated.…”

“…Indeed, autophagy is a process required for antigen presentation by dendritic cells and thus is intimately involved in adaptive immune responses (44), and the HIV-1 Nef protein was shown to inhibit autophagy (45,46). A possible interpretation is that autophagy may be induced in response to overexpression of ASP under the strong cytomegalovirus (CMV) promoter in transient-transfection assays (26,42). Therefore, it would be interesting to assess whether expression of ASP under its weaker natural antisense promoter located in the proviral 3= LTR also promotes autophagy (21,36).…”

“…Therefore, it would be interesting to assess whether expression of ASP under its weaker natural antisense promoter located in the proviral 3= LTR also promotes autophagy (21,36). The same studies elegantly demonstrated that ASP displays a propensity to form multimers and aggregates and that the N-terminal cysteine-rich region plays an important role in these events (26,42). The formation of aggregates may in part explain our observation that a highly hydrophobic protein such as ASP is able to reside in the nucleoplasm and that it is confined to limited areas of the nucleus rather than showing a diffused distribution.…”

Application of CRISPR/Cas9-Based Gene Editing in HIV-1/AIDS Therapy

“…HIV-1 is a retrovirus with an RNA genome of about 9.8 kb. The complete genome is flanked by two long terminal repeat (LTR) sequences and it encodes 10 viral proteins including gag, pol, vif, vpr, vpu, env, tat, rev, nef, and the antisense protein (ASP) (Cassan et al, 2016; Liu Z. et al, 2018), which have different functions in virus invasion and replication (Figure 1). Three main routes that spread HIV-1 are sex, intravenous injection, and vertical transmission (Hladik and McElrath, 2008; Cohen et al, 2011).…”