Figure 3: Sequence of 5′-UTR of lymphotropic HCV and cellular miR-122 levels are involved in lymphotropism.

(a) The putative structure of the 5′-UTR of SB-HCV RNA as determined by MFOLD program (computational secondary structure prediction software). The nucleotides in green boxes are those that differ between SB and JFH1 RNA. These nucleotides are located in stem-loops (IIIa and IIId) and bulge regions, which may recruit lymphocyte-specific factors. The nucleotides in red boxes are the first AUG (start codon). miR-122 seed-match sequences (red) and predicted interactions with miR-122 (green) are shown. (b) The HCV RNA titers in sh-RIGI-Raji cells transfected with various 5′-UTR mutants of SB RNA (*P<0.05, ANOVA, n=3). Error bars represent s.d. (c) The HCV RNA titers in sh-RIGI-Raji cells transfected with the various 5′-UTR mutants of JFH1 subgenomic replicon RNA (*P<0.05, ANOVA, n=3). Error bars represent s.d. (d) miR-122 expression levels in Raji cells transduced with lentivirus expressing shRIGI, sh-MDA5 or mir-122 (n=3). Error bars represent s.d. (e) Transduction of microRNA miR-122 enhances and stabilizes HCV replication in B lymphocytes. Effects of RIGI and miR-122 on lymphotropic HCV SB infection were examined using specific shRNA (n=3). Error bars represent s.d. (f) Human memory B cells have higher levels of miR-122 in clinical specimens. Cellular miR-122 levels were determined by qRT-PCR in quadruplicate in PBMCs from three different patients (n=3). Error bars represent s.d. (g) Analysis of HCV RNA in B cells versus miR-122 in B cells. The miR-122 levels of B cells from HCV patients were comparable to that of Raji cells. The miR-122 abundance is correlated with higher HCV RNA levels in memory B cells. Cellular HCV RNA levels were determined by qRT-PCR in nine different patient PBMCs. (h) Memory B cells from three patients were infected with HCV and cellular HCV RNA levels were determined by qRT-PCR in quadruplicate. (*P<0.05, Student t-test, n=3). Error bars represent s.d. (i) To determine if miR-122 sequestration reduced HCV replication in B cells, antagomir of miR-122 was transfected into human memory B cells isolated from HCV patients and examined for cellular levels of HCV RNA in B cells (*P<0.05, t-test, n=3). Error bars represent s.d.