In situ reactions of monoclonal antibodies with a viable mutant of murray valley encephalitis virus reveal an absence of dimeric NS1 protein

David C. Clark, Mario Lobigs, Eva Lee, Megan J. Howard, Kerri Clark, Bradley J. Blitvich, Roy A. Hall*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    64 Citations (Scopus)

    Abstract

    Studies on the NS1 protein of flaviviruses have concluded that formation of a stable homodimer is required for virus replication. However, previous work has reported that substitution of a conserved proline by leucine at residue 250 in NS1 of Kunjin virus (KUNV) eliminated dimerization, but allowed virus replication to continue. To assess whether this substitution has similar effects on other flaviviruses, it was introduced into an infectious clone of Murray Valley encephalitis virus (MVEV). Consistent with studies of KUNV, the mutant virus (MVEVNS1-250Leu) produced high levels of monomeric NS1 and the NS1 homodimer could not be detected. In contrast, wild-type MVEV cultures contained predominantly dimeric NS1. Retarded virus growth in Vero cells and loss of neuroinvasiveness for weanling mice revealed further similarities between MVEVNS1-250Leu and the corresponding KUNV mutant. To confirm that the lack of detection of dimeric NS1 in mutant virus samples was not due to denaturation of unstable dimers during Western blotting, a mAb (2E3) specific for the MVEV NS1 homodimer was produced. When NS1 protein as fixed in situ in mammalian and arthropod cells infected with wild-type or mutant virus, 2E3 reacted strongly with the former, but not the latter. These results confirmed that Pro250 in NS1 is important for dimerization and that substitution of this residue by leucine represents a conserved marker of attenuation for viruses of the Japanese encephalitis virus serocomplex. The inability to detect dimeric NS1 in supernatant or cell monolayers of cultures productively infected with mutant virus also suggests that dimerization of the protein may not be essential for virus replication.

    Original languageEnglish
    Pages (from-to)1175-1183
    Number of pages9
    JournalJournal of General Virology
    Volume88
    Issue number4
    DOIs
    Publication statusPublished - Apr 2007

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