Viral Determinants of miR-122-Independent C Virus Replication.

Hepatitis C virus (HCV) replication requires binding of the liver-speciﬁc microRNA (miRNA) miR-122 to two sites in the HCV 5 = untranslated region (UTR). Although we and others have shown that viral genetics impact the amount of active miR-122 required for replication, it is unclear if HCV can replicate in the complete absence of this miRNA. To probe the absolute requirements for miR-122 and the genetic basis for those requirements, we used clustered regularly interspaced short palindromic repeat (CRISPR) technology to knock out miR-122 in Huh-7.5 cells and reconstituted these knockout (KO) cells with either wild-type miR-122 or a mutated version of this miRNA. We then characterized the replication of the wild-type virus, as well as a mutated HCV bearing 5 = UTR substitutions to restore binding to the mutated miR-122, in miR-122 KO Huh-7.5 cells expressing no, wild-type, or mutated miR-122. We found that while replication was most efﬁcient when wild-type or mutated HCV was provided with the matched miR-122, inefﬁcient replication could be observed in cells expressing the mismatched miR-122 or no miR-122. We then selected viruses capable of replicating in cells expressing noncognate miR-122 RNAs. Unexpectedly, these viruses contained multiple mutations throughout their ﬁrst 42 nucleotides that would not be predicted to enhance binding of the provided miR-122. These mutations increased HCV RNA replication in cells expressing either the mismatched miR-122 or no miR-122. These data provide new evidence that HCV replication can occur independently of miR-122 and provide unexpected insights into how HCV genetics inﬂuence miR-122 requirements. of in the Western Hemisphere. HCV infection requires miR-122, which is expressed only in liver cells, and thus is one reason that replication of this virus occurs efﬁciently only in cells of hepatic origin. To understand how HCV genetics impact miR-122 usage, we knocked out miR-122 using clustered regularly interspaced short palindromic repeat (CRISPR) technology and adapted virus to replicate in the presence of noncognate miR-122 RNAs. In doing so, we identiﬁed viral mutations that allow replication in the complete absence of miR-122. This work provides new insights into how HCV genetics inﬂuence miR-122 requirements and proves that replication can occur without this miRNA, which has broad implications for how HCV tropism is maintained. 100 mg/ml streptomycin (Corning Life Sciences), and 5% fetal bovine serum (Gibco BRL Life Technologies). To stably express wild-type and p3-4 miR-122, miR-122 KO Huh-7.5 cells were transduced with TRIP lentiviral vectors encoding puromycin resistance linked to the miR-122 genomic locus as previously described (9) and selected with 1 mg/ml puromycin (Corning Life Sciences). CRISPR-mediated gene KO. Huh-7.5 cells were transduced to express a miR-122 sensor encom- passing a transcript encoding green ﬂuorescent protein and ﬁve 3 = UTR miR-122 target Sensor-expressing cells were then transiently transfected with expression plasmids encoding a human codon-optimized Cas9 protein from Streptococcus pyogenes (provided by George Church, Harvard University; Addgene plasmid number 41815) (19) and a miR-122-speciﬁc guide RNA targeting base 11 relative to the 5 = end of the mature miRNA (5 = AGAGCTGTGGAGTGTGACAA). Following passage for 2 weeks to allow the turnover of previously produced miR-122 RNAs, derepression of the reporter occurred in about 3% of the transfected cells, which were sorted with a ﬂuorescence-activated cell sorter (FACS), and single-cell clones were derived by dilution cloning.