Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys).
The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at a site in the viral RNA genome which is designated the primer-binding site (PBS). The HIV-1 PBS is an 18-nucleotide sequence that is complementary to the 3'-terminal 18 nucleotides of tRNA(3Lys), which is used as the primer for reverse transcription. All HIV-1 isolates sequenced to date contain a PBS complementary to tRNA(3Lys), suggesting that other cellular tRNAs might not function as primers for reverse transcription. To investigate this possibility, we have substituted the HIV-1 PBS with sequences predicted to be complementary to the 3'-terminal nucleotides of tRNA(1,2Lys), tRNA(Ile), and tRNA(His), which previous studies have identified to be packaged into HIV-1 virions along with tRNA(3Lys). We demonstrate that infectious viruses which utilized tRNA(1,2Lys), tRNA(Ile), and tRNA(His) in reverse transcription can be recovered. However, the appearances of viruses with PBSs complementary to these alternate tRNAs were delayed compared with the wild type. After extended in vitro culture, viruses containing the PBSs complementary to these different tRNAs reverted back to the wild-type PBS complementary to tRNA3(Lys). Furthermore, only the first 9 nucleotides of the 18 nucleotide PBSs were sufficient for HIV-1 to utilize the alternate tRNA primers in reverse transcription, demonstrating that HIV-1 does not require the complete 18-nucleotide PBS to utilize these tRNA primers for reverse transcription. These results suggest that factors other than complementarity between the PBS and the primer tRNA contribute to the selectivity of tRNA3(Lys) to initiate HIV-1 reverse transcription.
Wakefield
J K
JK
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
Wolf
A G
AG
Morrow
C D
CD
D000163:Acquired Immunodeficiency Syndrome / Q000097:blood; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D015483:HIV Antibodies / Q000097:blood; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012354:RNA, Transfer, His / Q000378:metabolism; D012355:RNA, Transfer, Ile / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D017931:DNA Primers; D015483:HIV Antibodies; D012346:RNA, Transfer, Amino Acyl; D012354:RNA, Transfer, His; D012355:RNA, Transfer, Ile; D014764:Viral Proteins; D012343:RNA, Transfer; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase