The initiation of HIV-1 reverse transcription utilizes a cellular tRNALys,3 as a primer. The 3′ terminal 18-nucleotides of the cellular tRNALys,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 tRNALys,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 tRNALys1,2 as a primer. An additional mutation in reverse transcriptase (RT), G490E, was found to facilitate the forced use of tRNALys1,2 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 tRNATrp. Previous studies from our laboratory have shown that these viruses are unstable and mutate to select tRNAMet or tRNALys1,2. Analysis of the replication of viruses with the U5 and PBS complementary to tRNATrp 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 tRNAMet as the primer for initiation of reverse transcription. In a previous study, we have described an HIV-1 that has been forced to utilize tRNALys1,2 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 tRNALys1,2 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. © 2006 Elsevier Inc. All rights reserved.