Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme

B Müller,T Restle,H Kühnel,R.S Goody

doi:10.1016/s0021-9258(18)98744-2

Abstract

A system for the expression of recombinant human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in Escherichia coli has been developed, which allows purification of the heterodimeric form of the enzyme as well as the separate purification of the two subunits. It is shown that equilibrium formation between monomeric and homodimeric forms of the recombinant 66- and 51-kDa subunits is considerably more rapid than in the case of the corresponding homodimeric forms of HIV-1 RT. In accordance with our previously published studies on HIV-1 RT (Restle, T., Müller, B., and Goody, R.S. (1990) J. Biol. Chem. 265, 8986-8988) RNA-dependent DNA polymerase activity of the HIV-2 RT preparations can be exactly correlated to their dimer content. No significant heterodimer formation can be observed upon coexpression of the 66-kDa subunit of HIV-2 RT with the 51-kDa subunit of HIV-1 RT in the same cell, indicating differences in the dimerization domains of the two proteins. Recombinant HIV-2 RT is not recognized by a set of 23 monoclonal antibodies raised against HIV-1 RT, although it shows weak cross-reactivity with sera from HIV-1-infected patients.

Highlights

Human immunodeficiency virus type2 (HIV-2) show fairlyhigh homology, but only -60% identity
To allow the preparation of significant amounts of the heterodimeric form, which, as for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, appears to be the form present in virus particles (DeVico et al, 1989) we constructed the coexpression vector pRT266/5s1hown in Fig. 1.Since the exact cleavage position resulting in formation of the 51-kDa subunit in virions remains to be determined, we decided to choose Phe-439as theC-terminal amino acid of the recombinant 51-kDa protein referring to the studies published on processing of the HIV-1 enzyme (Mizrahi et al, 1989; Graves et al, 1990)
The results presented here show that reverse transcriptase from HIV-2 can be expressed as a heterodimer in E. coli in direct analogy to theenzyme from HIV-1 (Muller et al, 1989)

Summary

Introduction

HIV-2 show fairlyhigh homology, but only -60% identity. it isof interest to characterize theenzyme from HIV-2 in order to study similarities adnidfferences to HIV-1 RTO. f particular interest to us were the crystallization properties, since the enzyme from HIV-1 does not readily give crystals suitable for x-ray diffraction studies. When the 66-kDa subunit of HIV-1 RT (p66) is expressed in E. coli, most of the protein is processed to the 66/51-kDa heterodimeric form during the purification procedure by contaminating bacterial proteases.

Results

Conclusion