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.

In addition to restricting access of RLRs, the MW is also predicted to function as a barrier to molecules required for virus replication including entry into the MW of newly synthesized viral proteins and the exiting of positive-strand RNA for translation. This segregation necessitates a transport mechanism capable of regulating traffic between compartments within the MW and the surrounding cytoplasm. We envision that a contributing factor to this barrier may be surrounding ER membranes, and we have previously proposed that NPCs positioned within these membranes could regulate transport of macromolecules into regions of the MW containing viral replication and assembly machinery [22, 37, 38] . Consistent with this idea, functional NPCs are detected in the ER (historically termed annulate lamellae) and we have reported that HCV-infected cells exhibit increased levels of NPC proteins in regions of the cytoplasm occupied by the MW [37, 75, 76] . We suggested that MW-associated NPCs would allow NLS-containing proteins, including several HCV proteins and nuclear factors important for viral infection, to move into and out of the MW. Here we have provided further evidence supporting a transport function for the MW-associated NPCs and soluble nuclear transport factors by demonstrating that RLRs, which are normally inhibited from accessing replication/ assembly complexes, colocalize with HCV proteins when they are tagged with an NLS sequence (Fig 6) . Additionally, we show that the colocalization between NLS-tagged RIG-I and HCV proteins can be inhibited by the addition of nuclear transport inhibitors (Fig 7) , further suggesting a role for the nuclear transport machinery in allowing access to the MW. Importantly, we also show that the addition of NLS sequences to active RLRs stimulates immune responses in HCV-infected cells and has an inhibitory affect on viral replication (Fig 8F) . These results are consistent with the NLS-RIG-I fusion protein gaining increased access to the MW and viral RNA. Thus, virus-induced recruitment of NPCs appears to contribute to selective transport into the MW and to the HCV immune evasion strategy.