Forced selection of tRNA Glu reveals the importance of two adenosine-rich RNA loops within the U5-PBS for SIV smmPBj replication

Abstract

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV-1) preferentially select and use tRNA Lys,3 as the primer for initiation of reverse transcription. Previous studies have shown that HIV-1 can be forced to use tRNA Glu 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 tRNA Glu. To examine the primer preference of SIV, mutations were made within the PBS of SIV smmPBj to be complementary to tRNA Glu. Analysis of the production of infectious virus revealed that SIV smmPBj with the PBS complementary to tRNA Glu retained approximately 80% infectivity of the wild type. However, modification of the U5 of SIV smmPBj to alter nucleotides to be complementary to the anticodon of tRNA Glu, in combination with the PBS complementary to tRNA Glu, drastically reduced the production of infectious SIV smmPBj to less than 1% that of wild type. The replication of SIV smmPBj with the PBS complementary to tRNA Glu was similar to that of the wild type virus, while the replication of SIV smmPBj with PBS and A-loop complementary to tRNA Glu was delayed compared to that of wild type virus. Analysis of the PBS regions revealed that the virus with the PBS complementary to tRNA Glu reverted quickly, within 4 days, to be complementary to tRNA Lys,3, while the virus with PBS and A-loop complementary to tRNA Glu retained the PBS for a longer time during in vitro culture although following extended replication both the A-loop and PBS of SIV smmPBj reverted to be complementary to tRNA Lys,3. RNA modeling of SIV smmPBj 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 SIV smmPBj, like HIV-1, preferentially utilizes tRNA Lys,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.