Structure and conformational dynamics of membrane-associated HIV-1 antisense protein ASP to aid viral entry

The National Institute of Allergy and Infectious Diseases awarded $251,249 to The Johns Hopkins University on July 8, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to characterize the structure and conformational dynamics of HIV-1 antisense protein (ASP), a membrane-associated viral protein implicated in viral entry.

The research will acquire the first experimental structural information about ASP in lipid membranes using continuous wave and pulse electron spin resonance (CW-ESR) studies with engineered tyrosine mutants to map the overall structure of membrane-proximal and membrane-distal regions of ASP. The project will also perform CW-ESR studies using spin-labeled ASP and envelope glycoprotein (gp120) reconstituted in lipid to determine whether and how the proximity of the two proteins influences ASP conformation. ASP has been detected in peripheral blood immune responses of people living with HIV-1 and in the envelope of infectious virions; knocking out ASP expression decreases viral entry. Despite these findings, ASP's structure has not been experimentally determined, and computational models diverge widely due to the absence of ASP orthologs or paralogs to guide prediction. Understanding ASP structure will enable structure-based studies of its role in viral entry.

Performance occurs in Baltimore, Maryland. The period of performance runs from July 8, 2026, through June 30, 2028.

Abstract

Pandemic HIV-1 group m strains encode an antisense protein (ASP) of ~189 amino acids and with a molecular weight ~20 kilodaltons (KDA). Expression of ASP is driven by a constitutive, TAT-independent negative sense promoter in the 3' long terminal repeat (LTR). The presence of cellular and humoral immune responses against ASP in peripheral blood of people living with HIV-1 (plwh) demonstrates that IT is expressed during HIV-1 infection in vivo. ASP is associated with various cellular compartments, including the plasma membrane where IT colocalizes with the GP120 envelope glycoprotein. ASP is also found in the envelope of infectious HIV-1 virions, which is consistent with our recent study showing that knocking out ASP expression decreases viral entry. Despite these intriguing findings, the structure of ASP has not been experimentally determined. Computational models of ASP topology and three-dimensional structure diverge widely because they utilize different prediction algorithms. Moreover, there are no ASP orthologs and paralogs to guide structure prediction. Thus, this lack of knowledge hinders structure-based studies into the role of ASP in viral entry. To overcome these deficiencies, this project will acquire the first experimental structural information about ASP in lipid. To this end, we will perform continuous wave and pulse electron spin resonance (CW-ESR) studies using engineered single and double tyrosine (y) mutants of membrane-residing ASP to map out the overall structure of membrane-proximal and -distal regions of ASP. Further, given the colocalization of membrane-associated ASP and GP120, we will also perform CW-ESR studies using spin-labeled ASP and ENV reconstituted in lipid environment to determine whether and how the proximity of the two proteins influences the conformation of ASP. In a parallel line of studies, we will investigate the topology of membrane-associated ASP and its role as an enhancer or facilitator of viral entry. These studies will utilize infectious HIV-1 strains that express deletion or substitutions mutants of ASP, or that lack ASP expression altogether. These "ASP-modified" infectious viral strains will be utilized to study the topology of membrane-associated ASP, its interaction with ENV, and its role in viral entry into primary CD4+ t cells. The design of ASP modifications will be based on the currently available knowledge of ASP topology, but they will also incorporate new conformational knowledge that will emerge from the structural studies in specific aim 1. Although the two aims are independent, new information emerging from each aim will feed into and support the other reciprocally. The proposed studies are of significance because they will provide key information about whether ASP is an enhancer or facilitator of the early steps of HIV-1 infection with targetable potential. The application presents several points of innovation both in the hypotheses proposed and in the technologies that will be utilized. The studies will be conducted by a team of investigators with proven records of relevant expertise.