Tisdale M, Schulze T, Larder B A, Moelling K
Molecular Sciences Department, Wellcome Research Laboratories, Beckenham, Kent, U.K.
J Gen Virol. 1991 Jan;72 ( Pt 1):59-66. doi: 10.1099/0022-1317-72-1-59.
The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.
人类免疫缺陷病毒（HIV）逆转录酶（RT）的C末端区域包含负责核糖核酸酶H（RNase H）活性的结构域。为了确定该RNase H结构域的重要性，通过氨基酸取代和特定缺失对在大肠杆菌中表达的重组RT的C末端区域进行了特定改变。通过滤膜分析和热失活动力学比较了针对蛋白质含量标准化的纯化野生型和突变型RT/RNase H蛋白的酶活性。组氨酸539突变为天冬酰胺的点突变产生了一种具有明显热不稳定RNase H功能的酶（失活增加9倍），而RT功能仅比野生型略微更不稳定（两倍）。第二个突变，组氨酸539突变为天冬氨酸，使两种酶活性受到相似程度的损害（4至5倍）。C末端缺失19个氨基酸（aa）（aa 540至560）和C末端截短21个aa（aa 540至560）降低了RT以及RNase H活性。一个缺失130个aa的酶没有RNase H活性且RT活性不足，无法进行失活研究。将两个突变体，即19个aa缺失突变体和组氨酸突变为天冬酰胺的突变体，引入前病毒HIV-1 DNA克隆中，以确定酶活性的变化，特别是RNase H活性的变化是否影响病毒感染性。两个突变体均无感染性，表明RT/RNase H蛋白的C末端19至21个氨基酸和组氨酸539对于HIV复制至关重要。这些结果与RNase H对于HIV-1感染性必不可少的假设一致。
