Identification and characterization of cell-adapted mutations in West Nile Virus and replicons



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Flavivirus persistence in cell culture is achieved by altering the interaction between the viral genome and the host cell. To identify some of the factors that contribute to a persistent West Nile virus (WNV) infection, WNV subgenomic replicon (WNR) genomes were modified to contain neomycin phosphotransferase (antibiotic-resistance) gene and cells that persistently harbored this WNR were selected for using G418. There were many changes to the genomes harvested from WNR-bearing cell cultures compared to the parental genome, perhaps the most striking was the prevalence of NS2A mutations. WNR and WNV genomes harboring these mutations replicated more poorly than wt WNR or WNV genomes in vitro. These NS2A mutant genomes, as well as a genome with a large deletion in the 3’ UTR, were highly attenuated Swiss-Webster outbred mice. Low levels of IFN were produced from cells infected with NS2A or 3’ UTR deletion WNR genomes compared to wt WNR genomes. Since the WNR and WNV cannot efficiently replicate, little stress is placed on the cell during replication, resulting in minimal engagement of the cell’s stress and apoptotic responses and a noncytopathic infection. As a result, little cytopathic effect and apoptotic death were observed in cells infected with NS2A or 3’ UTR deletion WNR mutants compared to cells infected with wt WNR genomes. This lack of death may have been attributed to low levels of CHOP, a pro-apoptotic protein that is induced during endoplasmic reticulum stress. Taken together, these data support the hypothesis that WNR and WNV genomes that replicate poorly do not efficiently produce signs of their replication that can be recognized by the cell and place little stress upon the cell, resulting in an attenuated noncytopathic, persistent infection.



West Nile virus, persistence, interferon, Flavivirus, ER stress, attenuation