Authors: Amanda Carlson
Mentors: Bradford Berges
Insitution: Brigham Young University
Human Immunodeficiency Virus (HIV) causes AIDS and is one of the most studied viruses in history. HIV is a retrovirus that has two copies of a single stranded RNA genome. While there is in-depth understanding of the virus and its pathogenesis, no completely effective treatment or vaccine exists.
One potential target for therapeutic treatment of HIV is Viral Protein R (Vpr). Vpr is a multi-functional accessory protein encoded by the HIV genome. While HIV is a quickly mutating virus, the vpr gene remains relatively conserved. Mutations in this protein dramatically impact the rate of AIDS progression compared to the wild type (WT) version of Vpr. The Vpr polymorphism R77Q is associated with the Long Term Non Progressor (LTNP) phenotype. Regular AIDS onset is 5-7 years for WT virus and 10 or more years for R77Q. These differences in AIDS progression have been observed in vivo by following people with HIV over time. We have successfully shown that R77Q activates G2 cell cycle arrest more efficiently than WT followed by apoptosis, a death mechanism with less inflammation compared to necrosis. While the molecular mechanism of Vpr-induced apoptosis is known, it is not yet determined why point mutations in Vpr are changing levels of apoptosis. With further experimentation, we have shown that R77Q has decreased expression of pro-inflammatory cytokines compared to WT virus, which may explain why it is associated with the LTNP phenotype.
The functions of Vpr come from binding and modifying cellular proteins and enzymes. The focus of our research is to determine what molecular interactions change between Vpr mutants to better understand the shifts in apoptotic levels. Vpr can be found intracellularly in the nucleus, cytoplasm, and mitochondria and extracellularly in secreted proteins and within virions. We will determine Vpr concentration in these various locations for both WT Vpr and the R77Q mutant, starting by measuring extracellular Vpr.
To quantify virion-associated Vpr, we have designed a research plan. We will use WT-Vpr plasmids tagged by GFP to create GFP-tagged plasmids with either WT, R77Q or null mutations using site-directed mutagenesis. We will use Sanger sequencing for confirmation of the proper Vpr mutations tagged by GFP. We will then digest the plasmid DNA, leaving only the Vpr-GFP component and use PCR to amplify the sequences. We will transfect null virus plasmid (NL4-3) and Vpr-GFP plasmids into HEK cells to package the null virus and Vpr-GFP plasmids together to create active HIV particles. Using these virus particles, we will infect Hut-78 cells for a short time to allow the virion to enter the cells. We will then measure GFP fluorescence via flow cytometry, allowing us to quantify virion Vpr. This will be run alongside a mock infection as a control. We hypothesize that differences in virion Vpr concentrations exist among Vpr mutants. Through these experiments, we aim to discover more about the role Vpr plays in cell death by apoptosis and contribute to the existing literature exploring the importance of Vpr in HIV-1.