ASSEMBLIN HOMOLOG OF HERPES-SIMPLEX VIRUS TYPE-1 RETAINS PROTEOLYTIC ACTIVITY WHEN EXPRESSED AS A RECOMBINANT 2-CHAIN ENZYME

The herpes simplex virus type 1 (HSV) maturational proteinase is synth esized as a precursor that undergoes two autoproteolytic cleavages; on e at its (M)aturational site, which eliminates its carboxyl ''tail,'' and a second at its (R)elease site, which separates the amino proteoly tic half of the precursor from its nonproteolytic carboxyl half. In cy tomegalovirus (CMV) the proteolytic half of the precursor, called asse mblin, undergoes a third cleavage at an (I)nternal site that converts it from a single-chain to a two-chain enzyme that retains activity. Th e HSV assemblin homolog has no I site and therefore does not form a co unterpart two-chain enzyme. In the work reported here we have cloned a nd expressed HSV sequences that encode mimics of the A(n) and A(c) sub units of two-chain CMV assemblin. We show that when these HSV sequence s are coexpressed in eukaryotic cells, the resulting subunits associat e spontaneously to form an active two-chain enzyme. We also show that the two-chain HSV enzyme, like the natural one-chain form, retains its marked preference for HSV over CMV substrates, and that intertypic re combinant two-chain assemblin (e.g., HSV A(n)/CMV A(c)) does not form because the cross-species subunits do not interact We conclude from th ese results that (i) there are not intrinsic structural differences in the HSV assemblin homolog that preclude its functioning as a CMV-like two-chain enzyme, (ii) the substrate selectivity shown by the single- chain HSV enzyme was not noticeably relaxed in the HSV two-chain mimic , and (iii) the interactive domains, through which the A(n) and A(c) p ortions of the single-chain enzymes associate, differ between HSV and CMV. (C) 1997 Academic Press