The mechanisms of Pol expression and assembly for human foamy virus
Baldwin, David Norris
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In this study, I have examined the role of Pol in the replication pathway of HFV. We discovered that unlike all known retroviruses, the Pol protein is expressed independently of Gag from a spliced mRNA, rather than as a Gag-Pol fusion protein. Subsequently, I compared the basic assembly strategies of foamy viruses, retroviruses and hepadnaviruses. Hepadnaviruses are the only other mammalian reverse transcriptase-encoding viruses which also express their Pol protein independently from their structural proteins. However, hepadnavirus assembly strategies are very different than those of retroviruses.I found that virus assembly with respect to Pol in HFV is more like retroviruses than hepadnaviruses. A pol deletion mutant was able to assemble particles which resemble wild type particles by electron microscopy. In addition, like other retroviruses, this pol deletion mutant was able to package wild type leves of genomic viral RNA. In contrast, however, I found that like hepadnaviruses, there is a requirement for the Env surface glycoproteins of HFV for virus release from the cell. In the absence of Env, capsids formed by HFV Gag could be seen by electron microscopy, and quantitative analysis of capsid formation indicated that there was no difference between wild type and the env deletion mutant, although no extracellular virus could be detected.The second part of this dissertation focused on mechanism by which HFV Pol is incorporated into particles. To study Pol incorporation I developed novel methods for detecting the Pol proteins in extracellular virions. By introducing a phosphorylation site for protein kinase A (PKA) into the integrase domain of Pol, I was able to immunoprecipitate and phosphorylate the Pol proteins from transfected cells, and gradient purified virions. Using this assay, I showed that proteolytic processing by the viral protease is not required for Pol incorporation. I was able to define domains of Pol and Gag which are important for Pol incorporation into particles. I found that the primary structure of Gag around the single cleavage site in Gag is not important, but that a region of about 8-kDa upstream is essential. I also found that the protease domain of Pol is essential for Pol incorporation.I also tested these domains of Gag and Pol in the yeast two-hybrid system and found that protein::protein interactions between Gag and PR could be detected. Since the truncation mutant of Gag which failed to package Pol was also lacking two of three basic regions known to be important for nucleic acid binding, I was interested in the ability of this virus to package RNA. I found that it was severely reduced for its ability to package RNA, beyond the limits of Pol detection using our PKA assay. I also tested a primer binding site (PBS) deletion mutant and found that it incorporated the Pol protein, implying that initiation of reverse transcription does not play a role in Pol assembly. While I found some evidence to suggest that protein::protein interactions between Gag and Pol are important for Pol incorporation, a role for RNA in this process cannot be ruled out from the work presented here.
- Microbiology