Zhang Z, Kang S M, LeBlanc A, Hajduk S L, Morrow C D
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
Virology. 1996 Dec 15;226(2):306-17. doi: 10.1006/viro.1996.0658.
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNA(Lys,3) as a primer and occurs at a site in the viral RNA genome, designated as the primer binding site (PBS), which is complementary to the 3'-terminal 18 nucleotides of tRNA(Lys,3). We previously described an HIV-1 virus [designated as HXB2(His-AC)], which contained a sequence within the U5 region complementary to the anticodon region of tRNA(His) in addition to a PBS complementary to the 3'-terminal 18 nucleotides of the tRNA(His). That virus maintained a PBS complementary to tRNA(His) after extended in vitro culture (Wakefield et al., J. Virol. 70, 966-975, 1996). In the present study, we report that subcloning a 200-base-pair DNA fragment encompassing the U5 and PBS regions from an integrated provirus of HXB2(His-AC) back into the wild-type genome (pHXB2) resulted in an infectious virus, designated as HXB2(His-AC-gac), which again stably maintained a PBS complementary to tRNA(His). DNA sequence analysis of the 200-base-pair region revealed only three nucleotide changes from HXB2(His-AC): a T-to-G change at nucleotide 174, a G-to-A change at nucleotide 181, and a T-to-C change at nucleotide 200. The new mutant virus replicated in CD4+ Sup T1 cells similarly to the wild-type virus. Comparison of the nucleotide sequence of nucleocapsid gene of the wild-type and HXB2 (His-AC-gac) virus revealed no differences. Although we found numerous mutations in the reverse transcriptase gene in proviral clones derived from HXB2 (His-AC-gac), no common mutations were found among the 13 clones examined. Comparison of the virion-associated tRNAs of HXB2(His-AC-gac) with those of the wild type revealed that both viruses incorporated a similar subset of cellular tRNAs, with tRNA(Lys,3) being the predominant tRNA found within virions. There was no selective enrichment for tRNA(His) within virions of HXB2(His-AC-gac) virus which selectively use tRNA(His) to initiate reverse transcription. The results of these studies suggest that the U5 and PBS regions in the viral RNA genome are important determinants for HXB2(His-AC) viruses in the selective use of tRNA(His) to initiate reverse transcription.
人类免疫缺陷病毒1型（HIV-1）基因组逆转录的起始需要细胞tRNA（Lys，3）作为引物，且发生在病毒RNA基因组中的一个位点，该位点被指定为引物结合位点（PBS），它与tRNA（Lys，3）的3'-末端18个核苷酸互补。我们之前描述过一种HIV-1病毒[命名为HXB2（His-AC）]，其U5区域内含有一段与tRNA（His）反密码子区域互补的序列，此外还有一个与tRNA（His）的3'-末端18个核苷酸互补的PBS。该病毒在体外长期培养后仍保持与tRNA（His）互补的PBS（韦克菲尔德等人，《病毒学杂志》70，966 - 975，1996）。在本研究中，我们报告将一个包含HXB2（His-AC）整合前病毒的U5和PBS区域的200碱基对DNA片段亚克隆回野生型基因组（pHXB2）中，产生了一种感染性病毒，命名为HXB2（His-AC-gac），它再次稳定地保持了与tRNA（His）互补的PBS。对该200碱基对区域的DNA序列分析显示，与HXB2（His-AC）相比仅存在三个核苷酸变化：第174位核苷酸由T变为G，第181位核苷酸由G变为A，第200位核苷酸由T变为C。这种新的突变病毒在CD4 + Sup T1细胞中的复制情况与野生型病毒相似。野生型和HXB2（His-AC-gac）病毒核衣壳基因的核苷酸序列比较未发现差异。尽管我们在源自HXB2（His-AC-gac）的前病毒克隆的逆转录酶基因中发现了许多突变，但在所检测的13个克隆中未发现共同突变。将HXB2（His-AC-gac）的病毒体相关tRNA与野生型的进行比较，结果显示两种病毒都掺入了类似的细胞tRNA子集，其中tRNA（Lys，3）是病毒体内发现的主要tRNA。在选择性使用tRNA（His）起始逆转录的HXB2（His-AC-gac）病毒的病毒体中，未发现tRNA（His）有选择性富集。这些研究结果表明，病毒RNA基因组中的U5和PBS区域是HXB2（His-AC）病毒选择性使用tRNA（His）起始逆转录的重要决定因素。
