The Impact of Multidideoxynucleoside Resistance-Conferring Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Polymerase Fidelity and Error Specificity

Variants of human immunodeficiency virus type 1 (HIV-1) that are highly resistant to a number of nucleoside analog drugs have been shown to develop in some patients receiving 2′,3′-dideoxy-3′-azidothymidine therapy in combination with 2′,3′-dideoxycytidine or 2′,3′-dideoxyinosine. The appearance, in the reverse transcriptase (RT), of the Q151M mutation in such variants precedes the sequential appearance of three or four additional mutations, resulting in a highly resistant virus. Three of the affected residues are proposed to lie in the vicinity of the template-primer in the three-dimensional structure of the HIV-1 RT–double-stranded DNA complex. The amino acid residue Q151 is thought to be very near the templating base. The nucleoside analog resistance mutations in the β9-β10 (M184V) and the β5a (E89G) strands of HIV-1 RT were previously shown to increase the fidelity of deoxynucleoside triphosphate insertion. Therefore, we have examined wild-type HIV-1(BH10) RT and two nucleoside analog-resistant variants, the Q151M and A62V/V75I/F77L/F116Y/Q151M (VILYM) RTs, for their overall forward mutation rates in an M13 gapped-duplex assay that utilizes lacZα as a reporter. The overall error rates for the wild-type, the Q151M, and the VILYM RTs were 4.5 × 10(−5), 4.0 × 10(−5), and 2.3 × 10(−5) per nucleotide, respectively. Although the mutant RTs displayed minimal decreases in the overall error rates compared to wild-type RT, the error specificities of both mutant RTs were altered. The Q151M RT mutant generated new hot spots, which were not observed for wild-type HIV-1 RT previously. The VILYM RT showed a marked reduction in error rate at two of the predominant mutational hot spots that have been observed for wild-type HIV-1 RT.

已有研究表明，在接受2′,3′-二脱氧-3′-叠氮胸苷（2′,3′-dideoxy-3′-azidothymidine）联合2′,3′-二脱氧胞苷（2′,3′-dideoxycytidine）或2′,3′-二脱氧肌苷（2′,3′-dideoxyinosine）治疗的部分患者体内，人类免疫缺陷病毒1型（human immunodeficiency virus type 1, HIV-1）可产生对多种核苷类似物药物（nucleoside analog drugs）高度耐药的变异株。此类变异株的逆转录酶（reverse transcriptase, RT）中先出现Q151M突变（Q151M mutation），随后依次出现3至4种额外突变，最终形成耐药性极强的病毒。其中3个受影响的氨基酸残基被认为位于HIV-1逆转录酶-双链DNA复合物（HIV-1 RT–double-stranded DNA complex）三维结构中模板-引物（template-primer）的邻近区域；氨基酸残基Q151被认为紧邻模板碱基（templating base）。此前已有研究证实，HIV-1逆转录酶的β9-β10（M184V）链与β5a（E89G）链上的核苷类似物耐药突变可提升脱氧核苷三磷酸（deoxynucleoside triphosphate）插入反应的保真度。因此，本研究采用以lacZα作为报告基因的M13缺口双链检测法（M13 gapped-duplex assay），检测了野生型HIV-1(BH10)逆转录酶以及两种核苷类似物耐药变异株（nucleoside analog-resistant variants）——Q151M和A62V/V75I/F77L/F116Y/Q151M（VILYM）逆转录酶的整体正向突变率（forward mutation rates）。野生型、Q151M及VILYM逆转录酶的整体错误率（overall error rates）分别为每核苷酸4.5×10^−5、4.0×10^−5和2.3×10^−5。尽管与野生型逆转录酶相比，两种突变型逆转录酶的整体错误率仅出现小幅下降，但二者的错误特异性（error specificities）均发生了改变：Q151M突变型逆转录酶产生了此前野生型HIV-1逆转录酶未观察到的新型突变热点（hot spots）；而VILYM逆转录酶在野生型HIV-1逆转录酶已发现的两类主要突变热点（predominant mutational hot spots）处的错误率出现了显著降低。