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

Abstract

The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target. IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.

A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients

“…As our results suggest that anti-ASP response is dynamic overtime, a deeper study of the relationship between viremia and the antibody response against ASP would require the analysis of these parameters in plasma sampled from patients at shorter temporal intervals. This could also indicate that the in vivo production of ASP occurs when viral production decreases, contrary to what was suggested by the study of Affram et al (2019), but in line with a previous study which suggested that ASP is rather expressed during the chronical phase than during the acute phase of the disease (Bet et al, 2015). Besides, we recorded an increase in ASPspecific antibody titers during the 2.5 years following enrollment in Pat #20, further suggesting that antibody response to ASP is developed after acute infection.…”

“…As suggested by the lack of detection of antibodies targeting ASP in the plasma of HIV-1 controllers in our study, a detectable level of viral replication might be needed to induce a humoral response to ASP. Interestingly, ASP was recently described at the membrane of viral particles released by chronically infected cells following their in vitro reactivation (Affram et al, 2019). If ASP is indeed present at the surface of viral particles in vivo, it might partially explain why we did not detect antibodies against ASP in patients displaying very low or undetectable viral loads.…”

Dynamically evolving novel overlapping gene as a factor in the SARS-CoV-2 pandemic

“…Such inconsistencies complicate research, because OLGs may play key roles in the emergence of new viruses. For example, in human immunodeficiency virus-1 (HIV-1), the novel OLG asp within env is actively expressed in human cells ( Affram et al, 2019 ) and is associated with the pandemic M group lineage ( Cassan et al, 2016 ).…”

Block and Lock HIV Cure Strategies to Control the Latent Reservoir

“…ASP RNA is derived from the 3′LTR and has been shown to recruit the polycomb group repressive complex 2 (PRC2) to the HIV-1 5′LTR promoter, resulting in repressive epigenetic modifications, i.e., increased H3K27me3 and reduced RNAPII occupancy at the promoter (Zapata et al, 2017 ). A recent study by Affram et al demonstrated that ASP is located in the nucleus of latent myeloid and lymphoid cell lines but translocates to the cytoplasm and cell surface upon stimulation with PMA (Affram et al, 2019 ). Further, ASP co-localized with gp120 on the cell surface and was observed in cell-free HIV-1 particles.…”