Hepatitis E virus (HEV) has a positive-sense RNA genome with a 5'-m7G cap. HEV open reading frame 1 (ORF1) encodes a polyprotein with multiple enzyme domains required for replication. HEV helicase is a nucleoside triphosphatase (NTPase) with the ability to unwind RNA duplexes in the 5'-to-3' direction. When incubated with 5'-[gamma-(32)P]RNA and 5'-[alpha-(32)P]RNA, HEV helicase released (32)P only from 5'-[gamma-(32)P]RNA, showing specificity for the gamma-beta-triphosphate bond. Removal of gamma-phosphate from the 5' end of the primary transcripts (pppRNA to ppRNA) by RNA triphosphatase is an essential step during cap formation. It is suggested that HEV employs the helicase to mediate the first step of 5' cap synthesis.