Forced selection of tRNA(Glu) reveals the importance of two adenosine-rich RNA loops within the U5-PBS for SIV(smmPBj) replication.
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.
Kelly
Maureen C
MC
Department of Cell Biology, University of Alabama, Birmingham, 802 Kaul Building, 720 20th Street South, Birmingham, AL 35294, USA.
Kosloff
Barry R
BR
Morrow
Casey D
CD
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D007963:Leukocytes, Mononuclear; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012352:RNA, Transfer, Glu / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D048348:Reverse Transcription; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000254:growth & development*; D014779:Virus Replication
D012352:RNA, Transfer, Glu; D012367:RNA, Viral