Identification of residues critical for the interferon antagonist function of Langat virus NS5 reveals a role for the RNA-dependent RNA polymerase domain

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton MT USA 59840

^* To whom correspondence should be addressed. Email: sbest{at}niaid.nih.gov.

	Abstract

All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis viruses) and Japanese encephalitis virus (JEV) use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355-735 (of 903) which lies within the RdRP domain. Furthermore, we identified two non-contiguous stretches of specific amino acids within the RdRP, 374-380 and 624-647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of anti-viral therapeutics that potentiate the action of IFN during infection.