The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

Our results indicate that the HCV-induced MW segregates activities associated with this structure, such as HCV replication and viral assembly, from cytosolic factors including ribosomes and RLRs. We have previously proposed that access of certain macromolecules to compartments within the MW is regulated by NPCs and nuclear transport factors, an observation consistent with both the presences of nuclear transport signals in multiple HCV proteins and our previous results showing a GFP-NLS-tagged reporter protein could enter regions of the cytoplasm occupied by the MW [37, 38] (see S4 Fig) . On the basis of these data, we hypothesized that placing an NLS on an RLR would overcome the selective barrier between the cytoplasm and the MW [37, 38] . For these experiments, an extensively studied NLS derived from the SV40 large T antigen (here referred to simply as the NLS) was used in the construction of various fusion proteins [59] . Constructs encoding GFP-tagged RIG-I-K270A, GFP-tagged NLS-RI-G-I-K270A, V5-tagged MDA5-I923V, or V5-tagged NLS-MDA5-I923V were transfected into uninfected or HCV-infected Huh7.5 cells, and the localization of each protein was compared to that of HCV NS5A. Similar to the K270A mutation in RIG-I, the I923V mutation in MDA5 inhibits MDA5-mediated activation of immune signalling pathways [60] . Consistent with our observations of MDA5 (Fig 1B) , the MDA5-I923V mutant was also observed outside cellular regions containing NS5A (Fig 6A) . In uninfected cells, both NLS-MDA5-I923V and NLS-RI-G-I-K270A exhibited an increased nuclear signal over those not containing an NLS sequence (MDA5-I923V and RIG-I-K270A), demonstrating that the NLS sequence is functional ( Fig 6A-6D) . Strikingly, when the NLS-MDA5-I923V and NLS-RIG-I-K270A proteins were examined in HCV-infected cells, the level of overlap between these RLRs and NS5A significantly increased compared to RLRs lacking the NLS (Fig 6A-6D) . Quantification of signal overlap using average Pearson's correlation coefficients of~20 cells revealed a significant increase in overlap between the fluorescent signals associated with NS5A and NLS-tagged RLRs when compared to NS5A and the untagged RLRs (Fig 6E) . These results led us to conclude that the addition of an NLS to RLRs, allowed the fusion protein to overcome the exclusion barrier normally preventing RLRs from entering the MW.