Jordan T. Becker, PhD

Position title: Sherer Laboratory

PhD in Cancer Biology Awarded Fall 2017

Dissertation Title. HIV-1 genomic RNA trafficking regulates virion assembly, incorporation of APOBEC3G, and interactions with host factors

Dissertation Summary. Subcellular localization is a critical determinant of mRNA utilization, stability, and interactions. Human immunodeficiency virus type 1 (HIV-1) becomes a cellular gene upon permanent integration into host chromosomal DNA. Manipulating HIV-1 RNAs may affect the location, rate, and fidelity of the viral life cycle. HIV-1 genomic RNAs (gRNAs) can serve as the mRNA for the Gag/Gag-Pol proteins that drive virion assembly and gRNA is the genetic material bound by Gag and packaged into virions. The nuclear export pathway of HIV-1 gRNA regulates cytoplasmic levels and localization of gRNA. gRNA trafficking regulates the kinetics of Gag expression and the onset of virion assembly. We sought to define the role of HIV-1 gRNA during the virion assembly process. Infectious HIV-1 virions efficiently (90%) package two copies of gRNA. What keeps Gag from driving the assembly of empty (gRNA-devoid) virions? If gRNA plays a role during virus particle assembly, it could be related to its role as the gag-pol mRNA. If HIV-1 gRNA controls the site of Gag synthesis and virus particle production, then artificially changing the subcellular localization of gRNA would change Gag localization and sites of assembly. We targeted Gag-encoding gRNAs to intracellular membranes and actin filaments. Retargeting gRNAs induced Gag accumulation at those sites and inhibited virion release. We reasoned that cellular and viral factors that specifically interact with HIV-1 gRNA and are incorporated into virions should traffic with gRNAs. Using cells stably-expressing a fluorescent version of cellular anti-viral restriction factor APOBEC3G, we observed that retargeting HIV-1 gRNAs, but not Gag, induced the accumulation of a majority of APOBEC3G to mistargeted locales. We observed that over 85% of virions contained gRNA and APOBEC3G. Less than 60% of virus particles derived from Gag-alone incorporated APOBEC3G. These results suggest HIV-1 gRNAs are a preferential means of APOBEC3G incorporation into virions. This work has broadened our understanding of HIV-1 gRNA trafficking during virion assembly. HIV-1 gRNA may regulate assembly by controlling the site of Gag synthesis as well as the site, size, and composition of HIV-1 assembling virions. Retargeting HIV-1 gRNA and inhibiting HIV-1 assembly could be used therapeutically.

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