The roles of the human immunodeficiency virus type 1 Pol protein and the primer binding site in the placement of primer tRNA(3Lys) onto viral genomic RNA.
Factors that modulate the placement of primer tRNA(3Lys) onto the viral RNA genome in human immunodeficiency virus type 1 (HIV-1) were investigated through analysis of reverse-transcribed products that are extended from the tRNA(3Lys) primer. Mutations were introduced into the HIV-1 pol gene to result in the appearance of a stop codon in the open reading frame of the reverse transcriptase (RT) gene. These constructs, BH10-RT1 and BH10-RT2, yielded viruses with truncated Pol proteins. Alternatively, we altered the sequences involved in frameshifting by generating the construct BH10-FS. With each of these mutated viruses, we found that the primer tRNA(3Lys) that was placed onto viral genomic RNA was present in an unextended state. In contrast, as expected, tRNA(3Lys) in the case of wild-type BH10 virus had been extended by 2 bases. Furthermore, the amount of tRNA(3Lys) that was placed onto viral RNA in mutated viruses was significantly less than that placed in the wild-type virus. We also generated a mutant within the polymerase-active site of RT (D185H) (Asp-->His) that eliminated RT polymerase activity. We found that the placement of primer tRNA(3Lys) onto viral genomic RNA was independent of enzyme function; however, the tRNA(3Lys) that was placed was present in an unextended state due to the loss of RT activity. In contrast, the elimination of protease activity through a D25A (Asp-->Ala) point mutation in the protease-active site (construct BH10-PR) did cause a drop in the efficiency of tRNA(3Lys) placement. In this situation, a proportion of the placed tRNA(3Lys) was found to be extended by 2 bases, although not to the extent found with wild-type virus (BH10), due to a decrease in RT activity associated with unprocessed Gag-Pol protein that could not be cleaved because of the loss of protease activity. We also investigated the role of the primer binding site (PBS) in the placement of tRNA(3Lys) through a series of 2-, 4-, and 8-nucleotide (nt) deletions at the 3' end of the PBS, i.e., BH10-PBS2, BH10-PBS4, and BH10-PBS8, respectively. In mutated viruses BH10-PBS2 and BH10-PBS4, the 2-base-extended form of tRNA(3Lys) was still detected. However, less primer tRNA(3Lys) was placed onto viral genomic RNA as more nucleotides were deleted until the percentage of placement seen with wild-type BH10 virus dropped to only 4% in the virus with 8 nt deleted (BH10-PBS8). Consistently, these mutated viruses possessed decreased initial replication capacity compared with that of the wild-type virus, with the extent of incapacity corresponding to the size of the deletion. However, after several days, an increase in replication potential was accompanied by a reversion to a wild-type PBS.
Liang
C
C
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada.
Rong
L
L
Morin
N
N
Cherry
E
E
Huang
Y
Y
Kleiman
L
L
Wainberg
M A
MA
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002461:Cell Line, Transformed; D017931:DNA Primers; D015683:Gene Products, gag / Q000378:metabolism; D015684:Gene Products, pol / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D011498:Protein Precursors / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D017384:Sequence Deletion; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015683:Gene Products, gag; D015684:Gene Products, pol; D011498:Protein Precursors; D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; C091950:pr160 gag-pol precursor protein, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase