Huang M, Zensen R, Cho M, Martin M A
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
J Virol. 1998 Mar;72(3):2047-54. doi: 10.1128/JVI.72.3.2047-2054.1998.
A temperature-sensitive (ts) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutant was generated by charged-cluster-to-alanine mutagenesis. The mutant virus, containing three charged residues within the RT finger domain changed to alanine (K64A, K66A, and D67A), replicated normally at 34.5 but not 39.5 degrees C. Quantitating virus particle production by p24 antigen capture or virion-associated RT activity and virus infectivity by the MAGI cell assay, we found that (i) mutant virions produced at the permissive temperature were indistinguishable from wild-type virus in assays performed at the nonpermissive temperature, suggesting that the ts mutation did not impair early steps in the virus replication cycle and that the mutant RT enzyme was not ts; and (ii) virus particle production in cells transfected with the ts mutant at the nonpermissive temperature was comparable to that of wild-type virus. However, the particle-associated RT activity and infectivity of mutant virions produced at the nonpermissive temperature were greatly reduced when assays were conducted at the permissive temperature. These results are consistent with an irreversible ts event affecting RT that occurs during virus particle production. Radioimmunoprecipitation analyses revealed that both p66 and p51 RT subunits were absent from mutant virions generated at 39.5 degrees C. The presence of normal levels of HIV-1 integrase in mutant particles produced at the nonpermissive temperature was inconsistent with defective Gag-Pol synthesis or Gag-Pol incorporation into progeny virions. Furthermore, wild-type levels of the mutant Pr160(gag-pol) were detected in virions produced at the nonpermissive temperature when the HIV-1 protease was inactivated by site-specific mutagenesis. Taken together, these results are most consistent with a ts defect affecting the degradation or aberrant processing of the mutated RT during its processing/maturation within nascent particles.
通过电荷簇到丙氨酸诱变产生了一种温度敏感（ts）的1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）突变体。该突变病毒在RT指状结构域内含有三个电荷残基转变为丙氨酸（K64A、K66A和D67A），在34.5℃时能正常复制，但在39.5℃时不能。通过p24抗原捕获或病毒体相关RT活性定量病毒颗粒产生，并通过MAGI细胞试验定量病毒感染性，我们发现：（i）在允许温度下产生的突变病毒体在非允许温度下进行的试验中与野生型病毒没有区别，这表明ts突变不会损害病毒复制周期的早期步骤，并且突变的RT酶不是温度敏感的；（ii）在非允许温度下用ts突变体转染的细胞中的病毒颗粒产生与野生型病毒相当。然而，当在允许温度下进行试验时，在非允许温度下产生的突变病毒体的颗粒相关RT活性和感染性大大降低。这些结果与病毒颗粒产生过程中影响RT的不可逆ts事件一致。放射免疫沉淀分析显示，在39.5℃产生的突变病毒体中不存在p66和p51 RT亚基。在非允许温度下产生的突变颗粒中存在正常水平的HIV-1整合酶，这与Gag-Pol合成缺陷或Gag-Pol掺入子代病毒体不一致。此外，当HIV-1蛋白酶通过位点特异性诱变失活时，在非允许温度下产生的病毒体中检测到野生型水平的突变Pr160（gag-pol）。综上所述，这些结果最符合一种温度敏感缺陷，该缺陷影响突变RT在新生颗粒内加工/成熟过程中的降解或异常加工。
