Schulze T, Nawrath M, Moelling K
Max-Planck-Institut für Molekulare Genetik, Berlin, Federal Republic of Germany.
Arch Virol. 1991;118(3-4):179-88. doi: 10.1007/BF01314028.
The reverse transcriptase/RNase H of HIV-1 is composed of a p66/p51 heterodimer when analyzed from virus particles. A recombinant reverse transcriptase (RT)/RNase H which after purification consisted mainly of p66 was analyzed as substrate of the purified recombinant HIV-1 protease p9 in vitro. The p66 protein if treated with the protease is processed to a stable p66/p51 heterodimer. A p15 protein is a prominent cleavage product which was identified as the carboxyterminal portion of p66 by means of a monoclonal antibody. It exhibits RNase H activity when tested by activated gel analysis. Presence of SDS during the incubation allowed complete degradation of p66 depending on the conditions, which indicates that conformation of a substrate is relevant for cleavage by the HIV-1 protease. A synthetic heptapeptide AET-FYVD derived from the region between RT and RNase H is cleaved efficiently in vitro by the HIV-1 protease at the F'Y junction, and may mimick a natural cleavage site. P66/p51 heterodimers exhibit higher RT and RNase H activities than p66 when renatured from polyacrylamide gels.
从病毒颗粒分析，HIV-1的逆转录酶/RNase H由p66/p51异二聚体组成。对一种纯化后主要由p66组成的重组逆转录酶（RT）/RNase H进行体外分析，将其作为纯化的重组HIV-1蛋白酶p9的底物。若用该蛋白酶处理p66蛋白，会加工成稳定的p66/p51异二聚体。一种p15蛋白是显著的切割产物，通过单克隆抗体鉴定其为p66的羧基末端部分。经活性凝胶分析测试，它具有RNase H活性。孵育过程中SDS的存在会根据条件使p66完全降解，这表明底物的构象与HIV-1蛋白酶的切割有关。源自RT和RNase H之间区域的合成七肽AET-FYVD在体外被HIV-1蛋白酶在F'Y连接处有效切割，可能模拟天然切割位点。当从聚丙烯酰胺凝胶复性时，p66/p51异二聚体比p66表现出更高的RT和RNase H活性。
