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.

Expression constructs for production of FLAG-tagged RIG-I (pEF-Tak-RIG-I and pEF-Tak-RIG-I(K270A)) were prepared by first preparing PCR products of the entire RIG-I open reading frame from pEF-BOS RIG-I and pEF-BOS RIG-I (K270A) [78] into pEF-Tak encoding tandem FLAG epitope sequences in frame with the RIG-I protein under control of the elongation factor 1 promoter. RIG-I K270 was first prepared by site-directed mutagenesis using the Q5 mutagenesis kit (New England Biolabs) of the WT RIG-I sequence. PCR and mutagenic primers, and cloning methods are available upon request. Expression constructs for the production of GFP-tagged RIG-I (pEGFP-RIG-I, pEGFP-RIG-I(K270A), pEGFP-NLS-RIG-I, and pEGFP-NLS-RIG-I(K270A)) were produced by PCR amplification of RIG-I from the pEF-Tak-RIG-I constructs followed by cloning into the pEGFP-C1 vector. The NLS sequence used was the well-characterized NLS derived from the SV40 large T antigen. Expression constructs for the production of MDA5 were produced by first reverse transcribing MDA5 mRNA from U937 cell extracts to form cDNA followed by PCR amplification using specific primers using the One-Step RT-PCR System (Invitrogen, 10928-042). Amplified MDA5 DNA was then cloned into a pcDNA3.1/nV5-DEST expression vector (Invitrogen, 12290010) using the gateway cloning system (Invitrogen). The MDA5(I923V) mutation was introduced by site-directed mutagenesis using the QuickChange Lightning mutagenesis kit (Agilent Technologies). The primes used for PCR amplification and site-directed mutagenesis are listed in S1 Table. Constructs were sequenced to confirm the proper mutation. Constructs were transfected into Huh7.5 cells using lipofectamine 2000 reagent (Invitrogen, 11668019) and expressed for 48 hours.