Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme

The two virus-specific reactions in the capping of alphavirus RNAs, catalyz ed by the replicase protein nsP1, are promising targets for developing viru s-specific inhibitors. In this report, we have studied the effect of over 5 0 cap analogs on the guanine-7-methyltransferase and guanylyltransferase ac tivities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Es cherichia coli and partially purified by flotation in a discontinuous sucro se gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent K-m values were 1.9 mM for GTP, 6.0 mu M for S-a denosyl-L-methionine and 170 mu M for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (3 5%), GppppG (50%) and less efficiently GpppA (12%), m(2)GTP (9%), and m(2,2 )GTP (25%), but not m(7)GppG. The most potent inhibitors for nsP1 methyltra nsferase were et(2)m(7)GMP (K-i value 42 mu M), m(2,7)GMP, (64 mu M), m(2,7 )GpppG (82 mu M), m2et7GMP (105 mu M), m(2)(2-phet)(7)GMP (194 mu M) and m( 2)GMP (386 mu M). Of these compounds, m(2)GMP, m(2)et(7)GMP and m(2)(2- phe t)(7)GMP showed competitive inhibition, whereas the others showed mixed typ e inhibition. All compounds that inhibited the methyltransferase activity i nhibited also the guanylyltransferase activity of nsP1. (C) 1999 Published by Elsevier Science B.V. All rights reserved.