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.

For analysis of intracellular RNA transcript levels, total RNA was extracted from cells using Trizol (Invitrogen, 15596018) and cDNA was synthesized using random primers (Invitrogen, 48190-011) and superscript II (Invitrogen, 18064014) according to the manufacturers specifications. Primers for qPCR were designed using Primer3 software and primer sequences are supplied in S1 Table. PCR efficiency for each primer was determined using the slope of a standard curve derived from qPCR analysis of cDNA serial dilutions. qPCR was done using a SYBR green super mix (Quanta, 95070-500) on a Stratagene Mx3005p real time PCR machine. To obtain the relative abundance of specific RNAs from each sample, cycle threshold (ct) values were corrected for the specific PCR efficiency of the primer, and normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT) transcript levels. For analysis of HCV RNA levels in subcellular fractions, RNA was isolated from 200 μl of the media obtained from infected cells using a High Pure Viral Nucleic Acid Kit (Roche, 11858874001). The cDNA was synthesized using superscript III (Invitrogen, 18080044) and an HCV specific primer (see HCV reverse primer sequence S1 Table) . qPCR was done using TaqMan Master Mix with HCV primers and labeled probe (S1 Table) .