title: The G490E mutation in reverse transcriptase does not impact tRNA primer selection by HIV-1 with altered PBS and A-loop.
abstract: The initiation of HIV-1 reverse transcription utilizes a cellular tRNA(Lys,3) as a primer. The 3' terminal 18-nucleotides of the cellular tRNA(Lys,3) are complementary to a region on the viral genome, designated as the primer binding site (PBS). Previous studies have shown that forcing HIV-1 to utilize alternative tRNA primers through alteration of the PBS results in viruses that revert to utilize tRNA(Lys,3) following in vitro replication. In some instances though, HIV-1 has been shown to select alternative tRNAs for initiation of reverse transcription if additional mutations upstream in the U5 region (A-loop) were made to be complementary to these alternative tRNAs. Recently, an HIV-1 has been described in which the U5 region distinct from the A-loop, designated as the primer activation site (PAS), was mutated in conjunction with the PBS to force the virus to use tRNA(Lys1,2) as a primer. An additional mutation in reverse transcriptase (RT), G490E, was found to facilitate the forced use of tRNA(Lys1,2) as the primer. In the current study, we have investigated the impact of the G490E mutation in RT on the selection and use of alternative primers by HIV-1. Viruses were first constructed in which the PBS and A-loop region were made complementary to tRNA(Trp). Previous studies from our laboratory have shown that these viruses are unstable and mutate to select tRNA(Met) or tRNA(Lys1,2). Analysis of the replication of viruses with the U5 and PBS complementary to tRNA(Trp) with or without the G490E mutation revealed no significant differences with respect to infectivity and viral growth in SupT1 or peripheral blood mononuclear cells (PBMC). In addition, the presence of the G490E mutation did not influence the capacity of this virus to revert to utilize tRNA(Met) as the primer for initiation of reverse transcription. In a previous study, we have described an HIV-1 that has been forced to utilize tRNA(Lys1,2) through mutations in the A-loop and PBS. The G490E RT mutation in this virus did not impact on the virus infectivity or growth in SupT1 or PBMC. We did not find a significant fitness advantage to viruses in which the A-loop and PBS were made complementary to tRNA(Lys1,2) that also contained the G490E mutation in RT. The results of these studies then establish that HIV-1 can be forced to use alternative primers through mutations in the U5 (PAS or A-loop) for certain tRNAs. Furthermore, for mutations in the A-loop and PBS, the RT does not play an important role in dictating the selection of the alternative primers to be used for initiation of reverse transcription.
authors.affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0024, USA.
authors.forename: Wenqin
authors.initials: W
authors.lastname: Xu
authors.forename: Casey D
authors.initials: CD
authors.lastname: Morrow
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; C034477:RNA primers; D012313:RNA; D012343:RNA, Transfer; D054303:HIV Reverse Transcriptase