Investigation of the C-Terminal Helix of HIV-1 Matrix: A Region Essential for Multiple Functions in the Viral Life Cycle: A Dissertation

Abstract

Since the first cases were reported over thirty years ago, great strides have been made to control disease progression in people living with HIV/AIDS. However, current estimates report that there are about 34 million individuals infected with HIV worldwide. Critical in the ongoing fight against this pandemic is the continuing development of highly active anti-retroviral therapies, ideally those with novel mechanisms of action. Currently, there are no medications approved for use that exploit the HIV-1 MA protein, despite its central role in multiple stages of the virus life cycle. This thesis sought to examine whether a highly conserved glutamate residue at position 99 in the understudied C-terminal helix of MA is required for HIV-1 replication. I characterized a panel of mutant viruses that contain different amino acid substitutions at this position using viral infectivity studies, virus-cell fusion assays, and immunoblotting. In doing so, I found that substitution of this glutamate with either a valine (E99V) or lysine (E99K) residue disrupted Env incorporation into nascent HIV particles, and abrogated their ability to fuse with target-cell membranes. In determining that the strain of HIV could affect the magnitude of E99V-associated defects, I identified a compensatory substitution at MA residue 84 that rescued both E99V- and E99K-associated impairments. I further characterized the MA E99V and E99K mutations by truncating HIV Env and pseudotyping with heterologous envelope proteins in an attempt to overcome the Env incorporation defect. Unexpectedly, I found that facilitating fusion at the plasma membrane was not sufficient to reverse the severe impairments in virus infectivity. Using quantitative PCR, I determined that an early post-entry step is disrupted in these particles that contain the E99V or E99K MA substitutions. However, allowing entry of mutant virus particles into cells through an endosomal route conferred a partial rescue in infectivity. As the characterization of this post-entry defect was limited by established virological methods, I designed a novel technique to analyze post-fusion events in retroviral infection. Thus, I present preliminary data regarding the development of a novel PCR-based assay that monitors trafficking of the viral reverse transcription complex (RTC) in an infected cell. The data presented in this thesis indicate that a single residue in MA, E99, has a previously unsuspected and key role in multiple facets of HIV-1 MA function. The pleiotropic defects that arise from specific substitutions of this amino acid implicate a hydrophobic pocket in MA in Env incorporation and an early post-entry function of the protein. These findings suggest that this understudied region of MA could be an important target in the development of a novel antiretroviral therapy.