PJ-1 The HIV-1 antisense protein ASP is a structural protein of the viral envelope
Authors: Affram Yvonne , Zapata Juan Carlos , Zhou Wei , Pazgier Marzena , Iglesias-Ussel Maria , Ray Krishanu , Latinovic Olga , Romerio Fabio 
Published: April 2019

Introduction:
The antisense strand of the HIV-1 genome encodes a 189-aa, highly hydrophobic protein (ASP) with no known homologs. Humoral and cellular immune responses against ASP in HIV-1 infected individuals demonstrate its expression invivo. However, whether ASP plays a role in viral replication is still unknown. We studied the expression and subcellular localization of ASP in various chronically infected cell lines. In addition, we investigated the impact of ASP expression on viral fitness.

Methods:
For intracellular and nuclear stainings, we used BD Cytofix/Cytoperm and eBioscience FoxP3 kits. Flow cytometry data were acquired on a Millipore Guava and analyzed with FlowJo. Confocal and super-resolution microscopy images were acquired on Zeiss LSM 800 and Nikon N-STORMTi-E microscopes, and analyzed with Zen Blue and Nikon Elements Software Version 3.1. For virion-capture assays, antibodies against ASP and gp120 were immobilized on Protein G-dynabeads. For Fluorescence Correlation Spectroscopy (FCS) we used an ISS Q2 confocal microscope and the data were analyzed with ISS VistaVision.

Results/Discussion:
We generated an in-house monoclonal antibody 324.6 against an epitope located between 2 putative transmembrane domains in the ASP protein. Using this antibody in flow cytometry and confocal microscopy, we found thatASP is expressed in the nuclei of chronically infected myeloid (U1andOM-10.1), and lymphoid (H9-IIIB, H9-CC, H9-MN, H9-RF, ACH2 and 8E5) celllines, but not their parental uninfected lines. However, ASP was not detectable neither on the cell surface or in the cytoplasm of infected cell lines. ASP expression was higher in cells cultured at low density compared to cells cultured at high density, suggesting that the rate of cell division may influence ASP expression levels. Confocal microscopy showed a polarized nuclear distribution of ASP, preferentially in areas of the nucleus containing actively transcribed chromatin. However, after reactivation of HIV-1 expression with PMA, we found that ASP translocated to the cytoplasm and to the cell membrane of all infected cell lines. In addition, the ability to detect ASP on the cell surface without permeabilization indicates that the ASP epitope recognized by324.6 antibody is extracellular. Confocal microscopy showed that ASP and gp120 co-localize on the cell surface of productively infected cells, and we determined that the Manders overlap coefficient is 78%. Super-resolution microscopy analyses provided further evidence of ASP and gp-120 co-localization. These results suggest that ASP might be incorporated in the membrane of budding virions upon their release from infected cells. Indeed, the anti-ASP 324.6 antibody was able to capture HIV-1 particles with an efficiency similar to the anti-gp120 VRC01 antibody. Moreover, FCS showed that the binding efficiency of 324.6 antibody to single virions in solution was ∼28%. Altogether, these 2 assays demonstrate the presence of ASP on the surface of mature HIV-1 particles released from infected cells. To investigate whether ASP expression impacts viral replication, we mutated single nucleotides in the HIV-1NL4-3 genome thus introducing early stop codons in the ASP open reading frame without affecting the amino acid sequence of Env encoded on the opposite strand. We found that these ASP-deficient viruses displayed a ∼50% reduction in replication rate compared to wildtype virus.

Conclusions:
Our results indicate that ASP is expressed in the nuclei of non-productively infected cell lines. Upon viral reactivation, ASP translocates to the surface where it co-localizes with gp120. Moreover, ASP is present on the membrane of viral particles, indicating that ASP is a structural protein in the envelope of mature HIV-1 virions. Further, ASP expression promotes viral replication. Overall, our results suggest that ASP may represent a new target for therapeutic or preventive vaccine.