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

PROJECT SUMMARY
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.