Last 20 aa of the West Nile virus NS1′ protein are responsible for its retention in cells and the formation of unique heat-stable dimers

Abstract

West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS19, a C-terminal extended product of NS1 generated as the result of a "1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS19, resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS19, or a combination of both, remains undetermined. Here, we showed that WNV NS19 formed a unique subpopulation of heat-and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS19-expressing plasmid showed that stability of NS19 dimers was linked to the penultimate 10 aa. To examine the role of NS19 heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS19 to create a WNV producing a truncated version of NS19 lacking the last 20 aa, but not affecting the PRF. NS19 protein produced by this mutant virus was secreted more efficiently than WT NS19, indicating that the sequence of the last 20 aa of NS19 was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNV KUN , suggesting that full-length NS19 was not essential for virus replication in vitro and for virulence in mice.

Japanese Encephalitis Virus NS1′ Protein Antagonizes Interferon Beta Production

“…For example, the NS4A protein of DENV sequesters MAVS to block IFN-I production, and WNV NS1 inhibits IFN-b responses by interacting with RIG-I and MDA5 (Dalrymple et al 2015;Zhang et al 2017). As observed for NS1 protein, NS1 0 protein has three forms, including a monomer (intracellular protein), membrane-bound protein (mNS1 0 ), and secreted protein (sNS1 0 ) (Young et al 2015). The region of NS1 0 that performs the IFN-I function will be explored in our next study.…”

Insect-Specific Flaviviruses: A Systematic Review of Their Discovery, Host Range, Mode of Transmission, Superinfection Exclusion Potential and Genomic Organization

“…Nevertheless, -1 PRF is utilized by various other flaviviruses. Apparently all viruses in the Japanese encephalitis (JE) serogroup, except for SLEV, utilize efficient -1 PRF to produce a larger NS1-related protein (NS1’) and to reduce synthesis of the 3'-encoded non-structural proteins relative to the proteins encoded upstream of the frameshift site [ 88 , 89 , 90 , 91 ]. -1 PRF has also been predicted to occur in many dISFs [ 87 ] (see Section 3 f) and in Wesselsbron and Sepik viruses [ 92 ].…”

Current Understanding of West Nile Virus Clinical Manifestations, Immune Responses, Neuroinvasion, and Immunotherapeutic Implications

“…There are three structural proteins (capsid [C], pre-membrane [PrM/M], and envelope [E]), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [4,5]. However, studies suggest that additional proteins may also be produced through ribosomal frameshifting [6,7]. While non-structural proteins play essential roles in the replication of viral genomic RNA, the structural proteins assemble the virion and mediate host receptor binding and viral entry.…”