Coupling Selection Of The HIV- 1 tRNA Primer Used For Reverse Transcription With Viral Translation And Encapsidation

HIV-1 belongs to the Retroviridae family. A hallmark of retroviral replication is the process of reverse transcription. Viral reverse transcriptase requires placement of a cellular tRNA primer onto the primer binding site (PBS) in order to initiate conversion of the viral RNA genome into a double stranded DNA copy. Mutation of the HIV-1 PBS forces the virus to utilize the non-cognate primer. Even though extensive studies have been done over the past 15 years, the mechanism of HIV-1 primer selection has yet to be completely elucidated. HIV-1 captures free tRNA during a yet undefined step in viral replication. However, our hypothesis that primer selection occurs at the level of translation is supported by data involving the HXB2yPhe virus. Moreover, encapsidation of viral genomic RNA has also been linked with translation. In order to further investigate this relationship, we constructed proviruses in which the PBS was altered to be complementary to either elongator methionine (tRNAMet(e)) (HXB2-Met(e))or initiator methionine (tRNAMet(i)) (HXB2-Met(i)). The tRNAs interact with a completely different set of host cell proteins and are involved in separate phases of translation. Transfection of HXB2-Met(i) produced fewer and less infectious virus. It was unable to productively infect SupT1 cells. However, it was noticed that this virus had an inadvertently ii introduced AUG codon in its PBS sequence. Therefore, a point mutation was introduced to alter the AUG to GUG (HXB2-Met(i) AG). This virus was able to productively infect SupT1 cells. Moreover, analysis of integrated proviral sequence revealed that it continuously selected tRNAMet(i) for use in reverse transcription. A model is presented which suggests that this acquisition of tRNA occurs as a result of ribosomal pausing at the gag-pol frameshift site. This model is supported by data that shows that codon usage in this region affects the stability of HIV-1 PBS mutants. Therefore, in addition to the PBS and A-loop, we have identified a third region of viral RNA that plays a role in primer selection. In order to further investigate the relative contribution of the PBS, A-loop and the frameshift region in HIV-1 primer selection we constructed a series of mutants. Three groups of viruses were separated based on PBS complementarity to tRNALys1,2, tRNALys3, or tRNAMet. Different combinations of A-loop and frameshift region mutations were introduced into the various proviral clones within the particular groups. The data obtained suggest that the frameshift region influences replication, although the specificity of primer is determined by the PBS and A-loop. Overall, the research presented in this dissertation has built upon previous findings and has linked primer selection, viral genomic RNA translation and encapidation.