Liang C, Rong L, Morin N, Cherry E, Huang Y, Kleiman L, Wainberg M A
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada.
J Virol. 1997 Dec;71(12):9075-86. doi: 10.1128/JVI.71.12.9075-9086.1997.
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.
通过对从tRNA(3Lys)引物延伸而来的逆转录产物进行分析，研究了调节人免疫缺陷病毒1型（HIV-1）中引物tRNA(3Lys)与病毒RNA基因组结合的因素。在HIV-1 pol基因中引入突变，导致逆转录酶（RT）基因的开放阅读框中出现终止密码子。这些构建体BH10-RT1和BH10-RT2产生了具有截短Pol蛋白的病毒。或者，我们通过构建BH10-FS改变了与移码有关的序列。对于每一种这些突变病毒，我们发现与病毒基因组RNA结合的引物tRNA(3Lys)处于未延伸状态。相比之下，正如预期的那样，野生型BH10病毒中的tRNA(3Lys)已经延伸了2个碱基。此外，突变病毒中与病毒RNA结合的tRNA(3Lys)的量明显少于野生型病毒中的量。我们还在RT的聚合酶活性位点产生了一个突变体（D185H）（天冬氨酸→组氨酸），该突变消除了RT聚合酶活性。我们发现引物tRNA(3Lys)与病毒基因组RNA的结合与酶功能无关；然而，由于RT活性丧失，结合的tRNA(3Lys)处于未延伸状态。相比之下，通过蛋白酶活性位点的D25A（天冬氨酸→丙氨酸）点突变（构建体BH10-PR）消除蛋白酶活性确实导致了tRNA(3Lys)结合效率的下降。在这种情况下，发现一部分结合的tRNA(3Lys)延伸了2个碱基，尽管由于与未加工的Gag-Pol蛋白相关的RT活性降低（由于蛋白酶活性丧失而无法切割），其延伸程度不如野生型病毒（BH10）。我们还通过在PBS的3'端进行一系列2、4和8个核苷酸（nt）的缺失，即分别为BH10-PBS2、BH10-PBS4和BH10-PBS8，研究了引物结合位点（PBS）在tRNA(3Lys)结合中的作用。在突变病毒BH10-PBS2和BH10-PBS4中，仍然检测到tRNA(3Lys)的2碱基延伸形式。然而，随着更多核苷酸被缺失，与病毒基因组RNA结合的引物tRNA(3Lys)越来越少，直到在缺失8 nt的病毒（BH10-PBS8）中，与野生型BH10病毒相比，结合百分比仅降至4%。一致的是，与野生型病毒相比，这些突变病毒的初始复制能力降低，丧失程度与缺失大小相对应。然而，几天后，复制潜力的增加伴随着向野生型PBS的回复。
