Gerondelis P, Archer R H, Palaniappan C, Reichman R C, Fay P J, Bambara R A, Demeter L M
Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
J Virol. 1999 Jul;73(7):5803-13. doi: 10.1128/JVI.73.7.5803-5813.1999.
The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5'-end-directed RNase H cleavage and slowed DNA 3'-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3'-end- and RNA 5'-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.
非核苷类逆转录酶（RT）抑制剂地拉韦啶（DLV）在体外可筛选出1型人类免疫缺陷病毒（HIV-1）RT突变P236L，该突变赋予对DLV的高水平耐药性，但对其他非核苷类逆转录酶抑制剂无耐药性。出乎意料的是，P236L在接受DLV治疗的患者分离出的HIV-1毒株中很少出现；K103N是在该情况下观察到的主要耐药突变。我们对来自含有P236L或K103N的pNL4-3的病毒在H9细胞和原代人外周血单个核细胞培养物中的复制适应性进行了表征，这两种细胞培养物同时感染了这两种突变体。在无DLV的情况下，野生型病毒的p24产生比任何一种突变体都更快且水平更高；相对于K103N，P236L的p24始终显示出两到三倍的降低。在低水平的DLV下，野生型病毒的生长受到严重抑制，K103N的复制效率比P236L高两到三倍。在高浓度的DLV下，P236L和K103N的复制均受到抑制。对含有K103N或P236L的重组RT进行了异聚RNA模板上的DNA聚合以及RNA-DNA杂交体的RNase H降解分析。两种突变体均未表现出聚合缺陷。与野生型RT相比，K103N表现出正常的RNA 5'-末端定向RNase H切割，而DNA 3'-末端定向RNase H切割减慢。P236L表现出DNA 3'-末端和RNA 5'-末端定向RNase H切割均减慢，这与其相对于K103N降低的复制效率一致。这些数据表明，非核苷类逆转录酶抑制剂耐药突变可导致RNase H切割效率降低这可能有助于降低HIV-1的复制适应性。
