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 against ASP in HIV-1 patients indicate that ASP is expressed ， 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 a polarized sub-nuclear distribution， preferentially in areas with low content of suppressive epigenetic marks. However， following treatment with phorbol 12-myristate 13-acetate (PM... More
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 against ASP in HIV-1 patients indicate that ASP is expressed ， 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 a polarized sub-nuclear 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 co-localize， suggesting that ASP might become incorporated in the membrane 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 anti-gp120 VRC01. Our studies indicate that ASP is an integral protein of the plasma membrane 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. The HIV-1 genome contains a gene expressed in the opposite or antisense direction of all other genes. The protein product of this antisense gene， called ASP， is poorly characterized and its role in viral replication remains still unknown. We provide evidence that the antisense protein， ASP of HIV-1 is found within the cell nucleus of unstimulated cells. In addition， we show that after PMA treatment ASP exits the nucleus and localizes on the cell membrane. Moreover， we demonstrated that ASP is present on the surface of viral particles. Altogether， our studies identify ASP as a new structural component of the HIV-1 virus， and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surface of both infected cells and viral particles might be exploited therapeutically.
