Author: Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.
Title: The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites Document date: 2016_2_10
ID: 1kuggdzj_11
Snippet: Active exclusion of proteins from the MW could represent an innate immune evasion strategy employed by the virus [8, 24, 37] . To test this hypothesis, we transfected uninfected or HCVinfected cells with constructs encoding FLAG-tagged RIG-I or V5-tagged MDA5 and visualized the cellular distribution of these RLRs by confocal immunofluorescence microscopy (Fig 1) . In uninfected cells, we observed a largely diffuse cytoplasmic localization for bot.....
Document: Active exclusion of proteins from the MW could represent an innate immune evasion strategy employed by the virus [8, 24, 37] . To test this hypothesis, we transfected uninfected or HCVinfected cells with constructs encoding FLAG-tagged RIG-I or V5-tagged MDA5 and visualized the cellular distribution of these RLRs by confocal immunofluorescence microscopy (Fig 1) . In uninfected cells, we observed a largely diffuse cytoplasmic localization for both RIG-I and MDA5, with small areas devoid of signal likely arising from the exclusion of the RLRs from vesicles (Fig 1A and 1B) . In HCV-infected cells, RIG-I and MDA5 were absent from larger regions of the cytoplasm and, importantly, these areas contained the majority of HCV proteins examined and viral dsRNA (Fig 1A and 1B bottom panels) . Pearson's correlation coefficients showed that there was a negative correlation between fluorescent signals observed for the HCV proteins/RNA and either RIG-I or MDA5, implying that the RLRs are restricted from accessing regions of the cytoplasm occupied by the MW. To further evaluate this apparent segregation, we examined the subcellular localization of the MDA5 reporter in Huh7 cells harbouring the JFH-1 subgenomic replicon. In replicon cells transfected with constructs coding for MDA5, we observed that MDA5 and viral proteins were also visible in distinct, largely non-overlapping regions of the cytoplasm similar to that seen in the HCV-infected cells (S1A Fig). These observations support the hypothesis that viral compartments inhibit entry of RLRs.
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