Nucleocapsid protein modulates the specificity of plus strand priming and recombination patterns in Human Immunodeficiency Virus
Jacob, Deena Thankam
DeStefano, Jeffrey J
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Replication in HIV (human immunodeficiency virus) occurs through reverse transcription in which the genomic single stranded RNA is copied into double stranded DNA. This process involves two priming events namely those of the minus and plus strand DNAs. The tRNA primer required to initiate the minus strand is carried by the virus into the host cell, while the plus strand primer is generated from a region of the genomic RNA called the polypurine tract (PPT). Results in this dissertation indicate a new role for HIV nucleocapsid protein (NC) in modulating the specificity of plus strand priming. For HIV, the central and 3′ (PPTs) are the major sites of plus strand initiation and other primers are rarely used. Using reconstituted in vitro assays, results showed that NC greatly reduced the efficiency of extension of non-PPT RNA primers, but not PPT. Extension assays in presence of mutant NCs show that the helix destabilization activity of NC and its ability to block the association of RT to non-PPT primers are responsible for the preferential extension of PPT in presence of NC. The effect of varying NC and Mg2+ concentrations on recombination during reverse transcription was also analyzed in this thesis. NC strongly influences the efficiency of recombination as well as the location where crossovers occurred. In contrast Mg2+ had a smaller effect on crossover locations. Both NC and Mg2+ influenced the level of pausing by RT during synthesis on RNA templates although NC's effect was more profound. At high NC concentrations, pausing was nearly eliminated even in locations with high predicted secondary structure. The results suggest that RT pausing may be limited during virus replication.