Intersection oftRNA biogenesis with primer selection by human immunodeficiency virus type I.

摘要

The initiation of retroviral reverse transcription requires a cellular tRNA primer that is selected from the intracellular milieu and placed at a site in the viral genome designated as the primer binding site (PBS). Although retroviruses have evolved to select a specific tRNA, genetic alteration of the PBS to be complementary to other tRNAs allows the selection of these tRNAs for replication. Little is known about how retroviruses select tRNAs for use in reverse transcription. This research focuses on the relationship between the availability and accessibility of the primer tRNA and the selection of the tRNA for the production of infectious virus.; Studies to elucidate the mechanism of tRNA selection from the intracellular milieu have been hampered due to the difficulties in manipulating the endogenous levels of tRNALys.3. A mutant HIV-1 with a PBS complementary to yeast tRNAPhe (psHIV-Phe) that relies on transfection of in vitro transcribed yeast tRNAPhe for infectivity has been previously described. To more accurately recapitulate the selection process, a cDNA was constructed for the intracellular expression of the yeast tRNA Phe and the capacity of the yeast tRNA to function within the normal biogenesis pathways within mammalian cells was established. Complementation of psHIV-Phe with endogenously expressed yeast tRNAPhe demonstrated that intracellular levels of primer tRNA can have a direct effect on HIV-1 infectivity and further support the role for PBS-tRNA complementarity in the selection process. To further investigate the features of primer selection with regard to the intracellular locale from which the primer is selected, yeast tRNAPhe mutants were designed such that they would be defective with respect to different steps in tRNA biogenesis. Yeast tRNA Phe mutants that were retained in the nucleus did not have the capacity to complement psHIV-Phe. Mutants that did not enter the channeled cycle of tRNAs during translation complemented psHIV-Phe, albeit, at levels lower than those observed for complementation with wild type yeast tRNAPhe. Collectively, our results demonstrate that capture of the tRNA primer by HIV-1 occurs post nuclear transport and support a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.