Forced selection of tRNAGlu reveals the importance of two adenosine-rich RNA loops within the U5-PBS for SIVsmmPBj replication

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV-1) preferentially select and use tRNALys,3 as the primer for initiation of reverse transcription. Previous studies have shown that HIV-1 can be forced to use tRNAGlu if mutations are made within the primer-binding site (PBS) and a region upstream, A-loop, to be complementary to the 3′-terminal 18 nucleotides and anticodon loop of tRNAGlu. To examine the primer preference of SIV, mutations were made within the PBS of SIVsmmPBj to be complementary to tRNAGlu. Analysis of the production of infectious virus revealed that SIVsmmPBj with the PBS complementary to tRNAGlu retained approximately 80% infectivity of the wild type. However, modification of the U5 of SIVsmmPBj to alter nucleotides to be complementary to the anticodon of tRNAGlu, in combination with the PBS complementary to tRNAGlu, drastically reduced the production of infectious SIVsmmPBj to less than 1% that of wild type. The replication of SIVsmmPBj with the PBS complementary to tRNAGlu was similar to that of the wild type virus, while the replication of SIVsmmPBj with PBS and A-loop complementary to tRNAGlu was delayed compared to that of wild type virus. Analysis of the PBS regions revealed that the virus with the PBS complementary to tRNAGlu reverted quickly, within 4 days, to be complementary to tRNALys,3, while the virus with PBS and A-loop complementary to tRNAGlu retained the PBS for a longer time during in vitro culture although following extended replication both the A-loop and PBS of SIVsmmPBj reverted to be complementary to tRNALys,3. RNA modeling of SIVsmmPBj U5-PBS by m-fold revealed two potential A-loop regions. Mutations in either A-loop drastically effected replication in human PBMC. Analysis of the A-loops following in vitro replication revealed that both reverted to the wild type sequence. The results of these studies demonstrate that SIVsmmPBj, like HIV-1, preferentially utilizes tRNALys,3 as a primer for reverse transcription for high level replication, but unlike HIV-1 selection may involve the use of two adenosine-rich loops.