Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2

B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5′-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts.

staining of sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel for supernatant, flow-through fraction, washing fractions and elution fractions purified from Sf9 cells infected with recombinant Baculovirus expressing truncated E2 protein (truncation of cytoplasmic domain). Recombinant HCV E2661 was purified by Baculovirus expression system by using of His-tag. (B) Immunoblot analysis by using of anti-His antibody to detect Histagged HCV E2 proteins purified from Baculovirus expression system. Monomeric and aggregated forms of E2 are indicated. (C) Lysates from Sf9 cells expressing HCV E2 proteins were separated by SDS-PAGE gel electrophoresis under non-reducing conditions. E2 was detected by immunoblotting with anti-E2 MAb. (D) Binding of E2 to Raji and HepG2-B7.2 cells in the presence or absence of a-B7.2 or isotype-matched control antibodies. The percentage of cells binding E2 was measured by FACS. Average values from two replicates are presented. Error bars represent standard deviation (n = 3, P<0.05). (E) Dose-response curve for E2 binding to Raji cells. Various amounts of E2 protein from genotypes 1a (H77 strain) and 2b (SB strain) were used in the binding experiment as in panel D.

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Infection and replication of HCV in B cells is not uncommon in HCV-infected patients 12

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HCV infection induces type III IFN, which is dependent on the MDA5 (IPS-1) pathway in CD8(+) DCs 42 and hepatocytes, leading to cytoplasmic antiviral protein expressions 16

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To generate pSB/JFH1/GND chimera virus construct carrying a mutation in the NS5B GDD motif, which 21 abolishes RNA polymerase activity, amino acid substitutions will be introduced by PCR-based site-directed mutagenesis in (MLVCGDDLVV) encompassing the GDD motif of NS5B, and amplified DNA fragments will be 23 analyzed by automated nucleotide sequencing by using an ABI 310 sequencer. GDD motif will be changed into 24 GND.

JFH-1 strain, SB strain from SB cells (spleen B cells), and SB/JFH1 chimera virus were used for in vitro
28 infection studies 18,22 . Genotype 1a/1b hybrid strain was previously described 23

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Negative strand-specific SB-HCV RNA was detected by using of a nested PCR method using 4-fold 46 serial dilution of RNA by using a recently established procedure previously described [25][26][27] . Viral RNA was purified with QIAmp Viral RNA Minikit (Qiagen). Negative strand-specific SB-HCV RNA were detected by use 48 of a nested polymerase chain reaction (PCR) method

RNA transfection
synthesized RNA was used for electroporation as previously described 22 . To determine whether HCV pseudo-particles are assembled in vitro, HCV envelope proteins were 2 expressed from a single polyprotein precursor and individually released in their respective cell compartments 3 on cleavage by cellular and viral proteases 28 . The expression vectors encoding the E1 and E2 glycoproteins 4 from several genotypes of HCV (1b, 2a or 2b) were generated by inserting a DNA fragment encoding the last 5 60 residues of HCV core and all of the E1 and E2 proteins into a non-packageable, CMV promoter-driven 6 expression construct pCDNA3.1 29 . To produce virus pseudo-types, HIV pseudo-types were generated by 7 cotransfection of 2.5  10 6 HEK293T cells were co-transfected in 10 cm-plates with a packaging-competent 8 plasmids encoding an envelope-defective HIV-1 proviral genome and the luciferase reporter in envelope-

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Exosome was isolated from plasma from HCV patients and healthy individuals by ExoQuick exosome 52 precipitation solution and centrifuged at 1,500g for 30 minutes from 100 μl of plasma after incubation at 4°C for committee approval was obtained in University of Southern California. The supernatant was denoted the 1 protein-rich fraction, whereas the pellet was denoted the exosome-rich fraction. The pellet (the exosome-rich 2 fraction) was washed twice with phosphate-buffered saline (PBS) and lysed with QIAzole (Qiagen) or lysed 3 with TRI reagent for total RNA or protein isolation. As the exosome precipitation solution precipitates up to 90 4 nm size particles, microparticles (≈200-1,000 nm) is in protein-rich fraction.

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Plasma MicroRNA Analysis 7 Re-suspend the UniSp6 RNA spike-in (from the Universal cDNA synthesis kit II,

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Calcium mobilization assay was performed as previously described 6 . After B cells (2 × 10 6 ) were incubated was measured with FACSCaliber and 405/495 nm emission ratios of IgM − B-cell were calculated.
Annexin V apoptosis assay was performed as previously described 6 . The cells were fixed in cold 70% ethanol 1 (0.5 ml) at 4 °C for 1 h. Memory B cells (2 × 10 6 ) were incubated with 1 μg/mL FLAG-tagged CD40L, 6 U/mL 2 IL-2 (R&D Systems) and 200 ng/mL IL-10 (R&D Systems) with 2 μg/mL mouse IgG 1 anti-FLAG antibody 3 (Alexis Biochemicals) in RPMI/10% fetal calf serum at 37°C for 0 and 6 hours and were washed with PBS and 4 resuspended. After incubation with annexin V-phycoerythrin at r.t., cells were stained with anti-CD27 FITC at 5 r.t. Flow cytometry was performed. Ratio of apoptosis was examined by a FACScan data acquisition for red 6 fluorescence.

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The secretion of IgM isotypes To test whether binding by HCV to B7.2 can downregulates B7.2-induced signaling pathways,

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immunoglobulin secretion assays were performed as previously described 6 . Resting B cells (50,000/well in 96-

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well round-bottom plates) were were purified from PBMC by negative selection and cultured for 6 days with 1 13 μg/mL FLAG-tagged CD40L, 6 U/mL IL-2 (R&D Systems) and 200 ng/mL IL-10 (R&D Systems) with 2 μg/mL  Inc.). The concentration of Abs is represented as nanograms/ml, as computed by using standard human IgM 31 isotypes.

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ANOVA analysis was used for multiple comparisons. Pearson's correlation test was performed for correlation