title: 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, NS1', 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 NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' 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 WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice.
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Lucy B
authors.initials: LB
authors.lastname: Young
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Ezequiel Balmori
authors.initials: EB
authors.lastname: Melian
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Yin Xiang
authors.initials: YX
authors.lastname: Setoh
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Young
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Khromykh
mesh_terms: D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D006863:Hydrogen-Ion Concentration; D051379:Mice; D055503:Protein Multimerization; D055550:Protein Stability; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D014901:West Nile Fever / Q000473:pathology / Q000821:virology
chemical_list: D017361:Viral Nonstructural Proteins; C531936:nonstructural protein 1, West Nile virus