Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome
replication and transcription. We previously reported several structural states
of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique
palm domain-based active site closure mechanism and proposed a six-state NAC
model including a hypothetical state representing translocation intermediates.
Using the RdRP from another human enterovirus, enterovirus 71, here we report
seven RdRP elongation complex structures derived from a crystal lattice that
allows three NAC events. These structures suggested a key order of events in
initial NTP binding and NTP-induced active site closure and revealed a bona fide
translocation intermediate featuring asymmetric movement of the template-product
duplex. Our work provides essential missing links in understanding NTP
recognition and translocation mechanisms in viral RdRPs and emphasizes the
uniqueness of the viral RdRPs compared with other processive polymerases.
