PROTEIN EXPRESSION AND PURIFICATION 3, 301-307 (19%) Comparative Analysis of Native and Cysteine-Deficient HIV-1 Reverse Transcriptase Meir Fischer,l Ruth Lifshitz, Tova Katz, Ilana Liefer, Hana Ben-Artzi, Marian Gorecki, Amos Panet, and Elisha Zeelon Biotechnology General (Israel) Ltd., Kiryat Weizmann, Rehovot, 76326 Israel Received March 6, 1992, and in revised form May 5, 1992 To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor spe- cies of 51,000 Da. Amino acid sequence analysis of the recombinant proteins revealed that the amino termini of the two major subunits are identical to that of the virion-derived enzyme. The two cysteinyl residues at positions 38 and 280 in the RT amino acid sequence were replaced by alanine in an attempt to elucidate the role of the sulfhydryl groups in RT enzyme activities, heterodimer formation, and intrasubunit linkage. The results reported here show that the two cysteinyls are dispensable and their absence in the amino acid se- quence of the reverse transcriptase does not affect DNA polymerase or ribonuclease H enzyme activities or the formation of heterodimer structures. Furthermore, in- hibitors of polymerase activity such as 3-azidothymi- dine triphosphate, dideoxythymidine triphosphate, and tetrahydroimidazo[4,5,1 -JK] [ 1,4]benzodiazepen- s(lH)-one are equally effective on the mutant contain- ing no cysteinyl residues and the wild-type enzyme. 0 1992 Academic Press, Inc. Human immunodeficiency virus 1 (HIV-l) reverse transcriptase (RT) has become the subject of intense study in pursuit of anti-HIV drug design. A large num- ber of drugs that are inhibitory to HIV RT, both in uiuo and in vitro, were described in recent years. The RT enzyme isolated from HIV virions is extremely difficult to obtain in larger quantities. The limited availability of purified HIV RT triggered a serious effort by a number ’ To whom correspondence should be addressed. 1046-5928/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. of investigators to clone and express the HIV-derived RT in Escherichia coli in large quantities (l-5). Al- though a great deal of information has been obtained by studying RT fusion proteins, they are less than optimal for analysis of the enzyme three-dimensional structure, active sites, and substrate specificities. Nonetheless, re- combinant RT that contains the authentic amino and carboxyl termini was obtained recently by coexpression of pal and HIV-l protease (5,6). These expression sys- tems have facilitated extensive genetic studies of RT and RNaseH (1,6-g). Analysis of deletions and site-di- rected mutants has indicated that the polymerase and RNaseH active sites are located at the amino and the carboxyl portions of the RT protein, respectively. How- ever, as most mutants are detrimental to both enzyme activities, the results suggest some overlapping of the two functional sites (5,6,10). Sequence analysis of HIV-l RT reveals only two cys- teinyl residues, at positions 38 and 280 of the protein (11). Cysteinyl residues are more abundant in the re- verse transcriptase of other retroviruses such as murine leukemia viruses (MuLV), which contain 7 residues, avian sarcoma leukemia viruses (ASLV), with 12 resi- dues, and human T-cell leukemia virus HTLV-1, which contains 7 cysteinyls (27). Indeed, the DNA polymerase activity of the latter enzymes is very sensitive to specific sulfhydryl agents (12,13). It has been suggested, there- fore, that cysteinyl residues are involved in the polymer- ase active site of MuLV and ASLV RTs. Hizi et al. (10) have recently reported that the two enzymatic activities of HIV-l RT responded differently to the sulfhydryl agent N-ethylmaleimide (NEM). DNA polymerase activity was resistant while RNaseH was inactivated by this agent. This observation pointed to the possible involvement of cysteinyl residues in the RNaseH active site of HIV-l RT. In the present report, we describe the physical and enzymatic properties of the recombinant HIV-l-derived 301