MA-RNA interactions in cells and their effect on HIV-1 assembly
Thornhill, Dishari
2020
Abstract
The HIV-1 structural polyprotein Gag drives the virus particle assembly specifically at the plasma membrane (PM). Gag binding to the PM is regulated by cellular factors including PM-specific phospholipid PI(4,5)P2 and RNAs, both of which bind the highly basic region (HBR) in the matrix domain (MA) of Gag. While it is well established that the interaction between the MA-HBR of Gag and the PM-enriched lipid PI(4,5)P2 is crucial for Gag localization to the PM, the role of MA-bound RNA in this process was less well defined. In this thesis, I examined the role of MA-bound RNA in the specific localization of HIV-1 Gag to the PM. The strategy that I employed was the comparison of Gag derivatives in which two of the eight basic amino acids in MA-HBR are substituted from Lys to Arg (KR) or from Lys to Thr (KT). Using a cell-based crosslinking assay, I determined the amount of RNA that is bound to the MA of the Gag derivatives in cells. Using microscopy, I determined the subcellular localization of these MA-HBR Gag mutants. Comparison of these data showed that an excellent correlation exists between the amount of MA-bound RNA in cells and the PM-specific localization of Gag. Overall, this study provided cell-based evidence supporting a model that MA-bound RNA prevents mislocalization of Gag to intracellular membranes, and in conjunction with PI(4,5)P2, helps Gag localize specifically to the PM. The predominant RNA species that binds MA-HBR in cells has been previously identified as tRNA with specific tRNAs bound to MA. It was not clear whether these tRNAs were selected by MA and if yes, then what were the determinants of selection of the specific tRNAs on MA. Using a Gag derivative that was designed to allow the capture of all the tRNAs bound to MA, and comparing the relative abundance of these tRNAs to their cellular abundances, I found that the tRNAs bound to MA were not just the highly abundant cellular tRNAs. These data suggest that specific tRNAs are selected by MA, and that the selection may be based on specific characteristics of tRNAs. The data also support that the tRNA sequence/structure as well as tRNA modifications are likely to contribute to the enrichment of preferred tRNAs to MA. I identified two tRNAs, SeCTCA and PheGAA, as being selected by MA in an MA-HBR dependent manner. These data support the role of the MA-HBR in selecting for specific tRNAs in cells. Comparisons of the relative abundances of SeCTCA and PheGAA between Gag derivatives that are either capable of membrane binding or not further suggest that these two tRNAs may regulate the membrane binding of Gag in cells in two different ways. SeCTCA could be a tRNA that is resistant to removal by PI(4,5)P2 and therefore prevents the binding of Gag to all cellular membranes, including the PM. PheGAA may be a tRNA that is sensitive to removal by PI(4,5)P2 and therefore allows Gag to bind specifically to the PM by preventing nonspecific binding of Gag to intracellular membranes. The analyses of MA-tRNA interactions could help inform the development of RNA aptamers with high binding affinity towards Gag such that they resistant to removal by PI(4,5)P2. Such RNA aptamers could serve as anti-retroviral agents that prevent the binding of Gag to the PM, and hence, block HIV-1 assembly.Subjects
HIV-1 Gag, tRNA, PI(4,5)P2, plasma membrane, subcellular localization, retrovirus assembly
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