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Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.
Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver, the fifth most common malignancy worldwide, and the third leading cause of cancer-related death, after cancers of lung and stomach (WHO Mortality Database [http://www.who.int/healthinfo/morttables/en/index.html]). The estimated incidence of new HCC cases is about 500,000 to 1,000,000 annually, with mortality of 600,000 cases per year on a global scale (12, 16, 17, 20, 24). Various risk factors for HCC include infection with hepatitis C virus (HCV) or hepatitis B virus (HBV), toxic exposures (alcohol and aflatoxins), metabolic disease (diabetes, nonalcoholic fatty liver disease, and hereditary hemochromatosis), and immune-related conditions such as primary biliary cirrhosis and autoimmune hepatitis (15).
The only established in vivo model for the study of HCV infection in an immunocompetent host is the chimpanzee (23). The inability of HCV to infect animals other than humans and chimpanzees has severely hampered efforts in developing a useful small animal model for the disease, specific antiviral therapies, and an effective vaccine against HCV-mediated liver cancer (18, 23).
In the United States, chronic HCV infection is the major etiological agent of liver cancer. Among individuals infected with HCV, approximately 80% develop chronic HCV infection, of which 20% will progress to cirrhosis, and 1 to 5% will progress to liver cancer (14). Genetic factors might affect the risk of liver cancer by modifying the susceptibility to HCV infection and viral clearance. Recent studies identified occludin (OCLN), an integral tight junction (TJ) protein, as one of the key factors for HCV entry into cells (8, 18). HCV infectivity was exclusively mediated by the second extracellular loop (EC2) of the OCLN MARVEL membrane-associating domain (18). This domain is found in proteins involved in lipid-rich membrane apposition events, such as cell fencing contacts and formation of vesicular particles (19). OCLN also has a large intracellular protein (ELL) domain, found in C-terminal parts of OCLN and in the ELL family of RNA polymerase II elongation factors (7), but its role in HCV infection is unclear.
We hypothesized that splicing diversity, generating multiple functionally distinct OCLN protein isoforms, might modulate susceptibility to HCV infection. Six splicing forms of OCLN and two distinct promoters, P1 and P2, have been described in cell lines (4, 5, 9, 10, 13). In the present study, we explored the splicing diversity of OCLN in normal human liver and observed variable expression of known and novel isoforms. Additionally, in vitro infectivity assays proved some of these forms to be nonpermissive for HCV infection. Our study suggests that naturally occurring splicing forms of OCLN might modify the outcome of HCV infection in humans.
Fresh-frozen liver tissue samples obtained from healthy liver donors (n = 15) were provided by the Liver Tissue Cell Distribution System under NIH contract N01-DK-7-0004/HHSN267200700004C after exemption by the Office of Human Subjects Research (OHSR) of the NIH (exempt 4539). Samples of total RNA from human tissues were purchased from Clontech and BioChain. Samples of total RNA from the NCI-60 panel of human cell lines from nine main types of cancers were provided by the Molecular Targets Team, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (DCTD), NCI/NIH. Additional cell lines were purchased from ATCC. Cells used for transfection and transduction experiments (HeLa, 293T, Huh-7.5, and 786-O) were maintained in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS).
Fresh-frozen liver samples were homogenized with TissueLyser (Qiagen). Total RNA was prepared with the mirVana kit (Ambion), and protein was prepared with radioimmunoprecipitation assay (RIPA) buffer (Pierce), complemented with a complete cocktail of proteinase inhibitors (Roche). RNA was quantified with NanoDrop (Thermo Scientific), and the integrity was determined with Bioanalyzer (Agilent). Protein concentration was determined by a standard bicinchoninic acid (BCA) protein assay (Pierce).
Protein lysates (~15 to 20 μg) were resolved on a 4 to 12% (for α-tubulin) or 10 to 20% Tris-glycine polyacrylamide gel (for OCLN), together with a prestained Rainbow marker (BenchMark prestained protein ladder; Invitrogen) and a protein marker (sc-2035; Santa Cruz). The gels were transferred to nitrocellulose membranes with iBlot (Invitrogen). After being blocked with 5% milk for 1 h at room temperature, the membranes were incubated with appropriate primary antibodies (anti-OCLN H00004950-B01 [MaxPab; Abnova, Taiwan] raised against the full-length human OCLN protein [522 amino acids], mouse anti-α-tubulin [ab-7291; Abcam], and rabbit anti-HaloTag [Promega]), all at 1:1,000 dilutions, and then incubated with appropriate secondary IgG-horseradish peroxidase (HRP) antibodies (sc-2305 donkey anti-rabbit and sc-2302 goat anti-mouse; Santa Cruz), at 1:10,000 dilutions. The immunostaining was detected with the ECL Plus Western blotting kit (GE Healthcare) and visualized on a G:Box imaging system (Syngene).
cDNA was synthesized from 1 μg of total RNA with random hexamers and the SuperScript III kit (Invitrogen) in a 20-μl reaction volume. cDNA samples were finally diluted with water to contain an equivalent of 5 ng/μl of total RNA. Expression assays were designed to uniquely measure expression of OCLN located at 68 Mb at 5q13.2 but not that of its partly duplicated copy located at 70 Mb. SYBR green assays were designed with Primer3 and NetPrimer software (http://frodo.wi.mit.edu/primer3/; http://www.premierbiosoft.com/netprimer/index.html). TaqMan expression assays were custom designed and manufactured by Applied Biosystems (see Table S2 in the supplemental material for a list of primers and assays). All assays were designed to cover exon-exon junctions, and specificity of assays was evaluated with an electronic PCR (ePCR) tool of the UCSC (http://genome.ucsc.edu/). Negative controls for all assays were represented by reactions that included 10 ng of genomic DNA and reactions that included total RNA but no reverse transcriptase (RT) enzyme (no RT control). Expression was quantified with the ABI Prism 7900HT sequence detection system (Applied Biosystems). The reactions included cDNA, 0.25 μl of 20× TaqMan or SYBR green gene expression assays, 2.5 μl of 2× gene expression master mix, or 2.5 μl of 2× Power SYBR buffer (Applied Biosystems). Three housekeeping genes, β2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin (ACTB), were tested as endogenous controls for expression in liver. Based on the level of interindividual variation in expression, B2M was selected as the best control in liver samples.
Full-length OCLN splicing forms were amplified from liver cDNA with cloning primers (see Table S2 in the supplemental material). The amplified products were cloned into the pFC8A (HaloTag) cytomegalovirus (CMV) Flexi vector (Promega) to create C-terminal HaloTag fusion proteins (18). The plasmids were verified by automated sequencing on a 3730 sequencer (Applied Biosystems) and by analysis with Sequencher 4.8 software (Gene Codes). Each of the recombinant OCLN clones was verified by transfection into the HeLa cell line, followed by Western blotting with the same anti-HaloTag antibodies later used for confocal imaging. All the clones produced proteins of expected sizes, ensuring reliability of OCLN-HaloTag protein detection by anti-HaloTag antibody.
cDNA fragments corresponding to complete open reading frames of six OCLN splice variants, cloned in the pFC8A (HaloTag) CMV Flexi vector, were transfected into HeLa cells using Lipofectamine 2000 reagent (Invitrogen). Nontransfected cells (Lipofectamine only) and cells transfected with empty green fluorescent protein (GFP)-tagged pIRES vector (Clontech) were used as negative controls. pIRES/GFP vector also served as a control for monitoring transfection efficiency.
HeLa cells grown in chamber slides were transiently transfected with Lipofectamine 2000 reagent and corresponding expression constructs. The cells were fixed for 20 min with 4% formaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X-100 in PBS for 5 min, blocked with 4% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h, and then incubated for 2 h with primary antibodies (Promega rabbit anti-HaloTag and Abcam ab-7291 mouse α-tubulin) at a 1:1,000 dilution. Then, the cells were incubated for 1 h with secondary antibodies (donkey anti-rabbit Alexa Fluor 594 and donkey anti-mouse Alexa Fluor 488, Invitrogen) at a 1:1,000 dilution. Slides were covered with mounting media (ProLong Gold antifade reagent with DAPI [4′,6-diamidino-2-phenylindole]; Invitrogen). Immunofluorescent images were obtained with a confocal laser scanning microscope (LSM 510 Meta; Carl Zeiss Jena GmbH, Germany). We used the HeLa cell line as a convenient and easily transfectable model for studies on subcellular localization of recombinant proteins. Endogenous expression of OCLN in HeLa cells is low, both on a mRNA level (our unpublished data) and on a protein level (Human Protein Atlas website [http://www.proteinatlas.org/]). Furthermore, like the 786-O cell line, the HeLa cell-derived cervical carcinoma line TZM was found to lack endogenous OCLN (25-fold less mRNA than that in Huh-7.5 cells ); therefore, it is unlikely that endogenous OCLN expression significantly interferes with expression of the recombinant OCLN protein or that expression is affected by the C-terminal HaloTag. The pattern of recombinant OCLN expression described by us is similar to that detected for endogenous OCLN expression in human tissues and cell lines (http://www.proteinatlas.org).
To generate a Gateway-compatible lentiviral vector for the expression of the OCLN splice variants fused to a red fluorescent protein (RFP), the pTagRFP-C vector (Evrogen) was modified as follows. Oligos encoding a C-terminal flexible linker (SGAGSAAGS) and a unique EcoRV site were inserted into the BspEI and SacII sites of pTagRFP-C, generating the pTagRFP-C-link plasmid. An EcoRV-flanked Gateway expression cassette (containing a chloramphenicol resistance gene and the ccdB suicide gene) from pLenti4.TO.V5-DEST (Invitrogen) was subcloned into pTagRFP-C-link to generate pTagRFP-C-link-DEST. The NdeI/SacII fragment from this vector was subcloned into corresponding sites of a pTRIP lentiviral expression construct (22, 25) to generate the pTRIP TagRFP-C-link-DEST construct. pENTR clones carrying sequences for the OCLN splice variants were used in LR reactions with the DEST vector to generate final lentiviral constructs for overexpression of RFP-tagged proteins.
HCVpp were generated as previously described (18). Briefly, 293T cells were cotransfected using FuGene 6 (Roche Applied Science), with plasmids carrying (i) a minimal HIV (pTRIP, CSGW) provirus encoding green fluorescent protein (GFP) or carrying another transgene; (ii) HIV Gag-Pol; and (iii) appropriate viral glycoproteins (HCV E1E2 or vesicular stomatitis virus glycoprotein [VSV-G]). To generate nonenveloped HCVpp (Env-pp), the glycoprotein vector was replaced with empty vector. Parallel transductions with pseudoparticles bearing the vesicular stomatitis virus glycoprotein (VSVGpp) were used for background normalization for HCVpp transductions, as previously described (18). Pseudoparticle-containing supernatants were collected at 24, 48, and 72 h, pooled, and filtered (0.45-μm pore size). Pseudoparticle transductions were performed in the presence of 4 μg ml−1 Polybrene. A minimum of 72 h elapsed between transduction and reporter gene quantification by flow cytometry or subsequent experiments.
For infectivity and coinfectivity assays with GFP reporter HCVpp, 2 × 104 cells were plated in 48-well plates. The next day, the cells were infected with HCVpp for 6 h. The media was changed, and cells were further cultured for 72 h prior to harvesting and fixation with 0.5% paraformaldehyde. GFP expression was quantified using a LSR 2 flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The background GFP signal from nonenveloped (NE) pseudoparticles was subtracted from the VSVGpp and HCVpp signals, and the HCVpp signal was normalized to VSVGpp infectivity [(HCVpp − NE)/(VSVG − NE)] and then normalized to HCVpp infectivity in Huh-7.5 cells to allow for cross-experimental comparison. Except where noted otherwise, results from the infection experiments are the means of at least two independently transduced populations. Errors bars represent the standard deviations of the means (see Fig. Fig.44 and and55).
The genomic region containing OCLN was analyzed with the use of the UCSC Genome Bioinformatics browser (http://genome.ucsc.edu/). The sequences of OCLN were analyzed with following tools: Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/), BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and Protein Calculator v3.3 (http://www.scripps.edu/~cdputnam/protcalc.html).
mRNA expression of all assays was measured in technical duplicates. Expression values were averaged and normalized by the expression values of B2M, measured in separate reactions but with the same cDNA preparations. Expression values were tested for normality of distribution and analyzed according to the threshold cycle (ΔΔCT) method of relative quantification (1). Statistical analyses were performed with Microsoft Office Excel and SPSS 16.0 software.
Human OCLN maps to two different genomic locations, as follows: a full-length copy is located at 68 Mb on chromosome 5q13.2, while a partially duplicated truncated copy of OCLN, referred to as the OCLN pseudogene (LOC647859), is located 1.5 Mb telomeric of that at 70 Mb (Fig. (Fig.1).1). Truncated OCLN is included in a 500-kb inverted duplicated region that contains four genes. The duplicated region of OCLN begins within exon 5, continues to the end of the gene, and has an open reading frame for a truncated OCLN protein isoform of 14.3 kDa. This isoform lacks the membrane-associating MARVEL domain (18) but retains the intracellular ELL domain found in OCLN and in the ELL family of RNA polymerase II elongation factors (3, 19, 21).
We evaluated OCLN mRNA expression in liver tissue samples obtained from 15 healthy liver donors and detected expression of transcripts from both of the promoters (P1 and P2) (10). We also detected expression of the previously described splicing form with deletion of exon 4 (OCLN-ex4del) (10). However, we did not observe expression of other splicing forms previously detected in cell lines with deletion of exon 9, deletion of exons 4 through 7, and partial deletion of exon 6 (data not shown) (5, 9). To fully explore the splicing diversity of OCLN in normal human liver, we cloned all transcripts from the exon 2 translation start site (used in transcripts from both promoters) to the stop codon within terminal exon 9. In addition to the wild-type form (WT-OCLN), we observed several novel alternative splicing forms with distinct in-frame deletions resulting in truncated protein fragments. The potential use of internal translation start sites within exon 4 or 5 will result in N-terminally truncated proteins without the MARVEL domain but with the ELL domain (Table (Table1).1). Analysis of OCLN protein expression in normal liver samples showed several bands on the Western blot (Fig. (Fig.2).2). The strongest bands may correspond to the WT-OCLN form (59.1 kDa), the form with extension of exon 7 (OCLN-ex7ext; 54.1 kDa), and the form with in-frame deletion of exon 4 (OCLN-ex4del; 52.7 kDa). Additionally, we observed several minor forms that may correspond to other alternative forms of OCLN (Table (Table11 and Fig. Fig.1B1B and and2).2). Next, we quantified mRNA expression levels of all splicing forms in individual liver samples. Expression of OCLN transcripts, normalized to expression of the endogenous control β2-microglubilin (B2M) or to expression of WT-OCLN, strongly differed between individuals (Table (Table22).
Expression constructs carrying full-length cDNAs for WT-OCLN and several splicing forms were transiently transfected into HeLa cells to determine the subcellular localization of the corresponding proteins. The two forms containing entire MARVEL domains (WT-OCLN and OCLN-ex7ext) were expressed predominantly on the cell membrane, whereas forms lacking the entire or part of the MARVEL domain were observed predominantly in the cytoplasm (Fig. (Fig.33).
Next, we tested which of the OCLN protein isoforms are capable of mediating HCV entry (Fig. (Fig.4).4). The human renal carcinoma cell line 786-O is naturally resistant to HCV entry due to low endogenous expression of OCLN (see Table S1 in the supplemental material) (18). In agreement with previous data (18), overexpression of proteins with intact MARVEL domains (WT-OCLN and OCLN-ex7ext) in 786-O cells substantially enhanced infectivity by HCV pseudoparticles (HCVpp). In contrast, other protein forms with modified MARVEL domains were not capable of restoring HCV entry into 786-O cells, despite similar expression levels of recombinant OCLN protein (as measured by TagRFP fluorescence) (data not shown). This is in concordance with the expected role of the MARVEL domain in HCV entry (18) and in defining subcellular localization patterns (Fig. (Fig.3).3). Therefore, the WT-OCLN and OCLN-ex7ext forms were designated permissive forms, while all other OCLN forms were designated nonpermissive for HCV infection.
In a panel of human tissues, mRNA expression of WT-OCLN was found in all samples tested, except whole-blood and purified peripheral blood monocytes. Relative expression (compared to that of liver) of the HCV-permissive and -nonpermissive forms differed strongly among tissues (Table (Table3).3). For example, in the pancreas, the expression level of permissive WT-OCLN was higher by 1.6-fold, while expression levels of several nonpermissive forms were higher by 23-fold (OCLN-ex4del), 13-fold (OCLN-ex3-4del), and 3.5-fold (OCLN-ex3pdel). Similarly, in the colon, the expression level of the permissive WT-OCLN form was higher by 2-fold, while expression levels of nonpermissive forms were higher by 9.5-fold (OCLN-ex4del) and 2.2-fold (OCLN-ex3-4del).
We tested mRNA expression of WT-OCLN and 5 alternative splicing forms of OCLN in 82 human cell lines (NCI-60 panel and additional cell lines) (see Table S1 in the supplemental material). In general, the highest level of expression of WT-OCLN was observed in cell lines derived from the colon and liver, while little or no expression was observed in melanoma, kidney, and blood-derived cell lines. Interestingly, no expression of WT-OCLN was detected in some blood-derived cell lines, including Daudi, Raji, CCRF-CEM, RPMI 8226, SR, and bone marrow K562 cell lines, while high expression of several forms, including WT-OCLN, was observed in other blood-derived cell lines, including the monocytic histiocytic lymphoma cell line U-937; a human T-cell line immortalized with human T-cell lymphotropic virus type 1 (HTLV-1), MT-2; and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (B cells) (see Table S1 in the supplemental material).
It is conceivable that higher levels of relative expression of OCLN splice variants that do not efficiently support HCV entry exert a dominant-negative effect on cells expressing wild-type OCLN at lower levels. To experimentally address this possibility, we expressed N-terminally tagged wild-type OCLN (Venus/yellow fluorescent protein [YFP]) and/or OCLN splice variants that do (-ex7ext) or do not (-ex4del) support HCVpp entry into 786-O cells (Fig. (Fig.5).5). We gated on cells expressing high and low levels of the respective HCV entry factor variants (as indicated by the mean fluorescence intensities [MFI] for different cell populations) and quantified the relative HCVpp infectivity. An approximately 5-fold-higher level of expression of OCLN-ex4del led to a <2-fold reduction in HCVpp entry infectivity in 786-O cells that simultaneously expressed wild-type OCLN (Fig. (Fig.5,5, second row) compared to that of the population expressing the two variants in inverted ratios (first row). However, in the latter population, HCVpp permissiveness was still comparable to that of cells expressing only wild-type OCLN (Fig. (Fig.5,5, ninth row) or coexpressing wild-type OCLN and OCLN-ex7ext (fifth and sixth rows). Thus, we conclude that OCLN splice variants that do not support HCV entry do not render otherwise permissive cells incapable of taking up HCV in vitro.
HCV infection is an important risk factor for the development of chronic liver disease and liver cancer. OCLN was recently identified as a major cell entry factor for HCV (8, 18). Here, we have extensively explored the natural splicing diversity of OCLN in normal human liver and shown that OCLN protein isoforms differ in their ability to support HCV infection in vitro.
OCLN is an integral TJ protein. TJs connect neighboring cells, create the primary barrier to the diffusion of solutes, and polarize epithelial and endothelial cells by separation of the apical and basolateral plasma membrane domains. Several proteins, including the HCV entry factors OCLN and CLDN-1, can be found in TJ complexes. However, it seems that OCLN is not an obligatory part of these complexes, as TJs can be formed in its absence (2). Furthermore, in a mouse model in which deletion of OCLN exon 3 resulted in the creation of a splicing form similar to the one we identified in human liver (OCLN-ex3del), the structure and function of TJs were not affected (2). The animals did not exhibit any clear phenotypes except chronic gastritis, abnormalities in the testes, salivary glands, and bones, and progressive accumulation of mineral deposits in the brains of aged animals (2). The resulting OCLN-ex3del protein is N-terminally truncated and lacks the extracellular MARVEL domain but retains the intracellular ELL domain (7). In agreement with the reported absence of expression of this form at the TJ (2), we did not observe expression of the human recombinant protein encoded by this form on the cell membrane. In in vitro infectivity assays, only OCLN protein forms with intact MARVEL domains (both extracellular loops encoded by exon 3 and the second transmembrane domain encoded by exon 4) could support HCV infection. Thus, we can conclude that the MARVEL domain is critical for OCLN function both as a TJ protein and as a receptor for HCV entry, but it might not be essential for other cell functions. The only splicing form identified in human liver that retains the MARVEL domain but lacks a part of the ELL domain (the OCLN-ex7ext splicing form) encodes a protein that localizes to the cell membrane and is permissive for in vitro HCV infection. It is possible, however, that this form was not as efficient in supporting infectivity as WT-OCLN due to the partial truncation of the intracellular ELL domain (Fig. (Fig.5).5). Interestingly, OCLN proteins of humans and chimpanzees, the only species susceptible to HCV infection, are 100% identical, while OCLN of mice, a genus resistant to HCV infection, shares only 89% identity with human protein.
Among different human cells types, only hepatocytes are permissive to HCV infection and capable of supporting viral replication (11). Possible explanations for this specificity are the following: HCV infects only polarized cells (however, many epithelial polarized cell types are not infected by HCV), and some components required for infection (OCLN, CLDN-1, CD81, and SR-B1) are absent in cells that resist infection. Particularly intriguing is why HCV does not infect blood cells (11), the primary contact sites for HCV in the human body. Our results suggest that tissue and cell-specific relative expression of the permissive and nonpermissive splicing forms of OCLN might be a critical factor determining the susceptibility to HCV infection. However, our in vitro infectivity assays that included different combinations of coexpressed OCLN constructs did not indicate that HCV entry is markedly repressed in the presence of OCLN-ex4del, a splice variant that does not support HCV entry. OCLN-ex4del retains the extracellular loops but lacks the second transmembrane domain (encoded by exon 4). Nonetheless, the relative HCV entry efficacy could be additionally influenced by the abundance of multiple different splice forms that might sequester potentially limiting adaptor molecules as well as cell polarity and result in more confined subcellular spatial distribution of the respective OCLN variants.
We did not observe mRNA expression of WT-OCLN or alternative splicing forms in human peripheral blood mononuclear cells, human-purified monocytes, or multiple blood-derived cell lines. At the same time, expression was high in other blood-derived cell lines, including the monocytic U-937 cell line, Epstein-Barr virus (EBV)-transformed human B-lymphocytes, and HTLV-1-transformed MT-2 cell line (T cells). Interestingly, it has been reported that both U-937 and MT2 cells may support some level of HCV infection. Such a discrepancy in OCLN expression and HCV infectivity in primary blood cells and transformed blood cells warrants future studies.
Genetic variants near the IL28B (interleukin 28B) gene encoding interferon-γ3 are associated with response to alpha interferon-ribavirin combination therapy for HCV infection (14) and spontaneous clearance of HCV (14). Genetic factors that regulate relative expression of OCLN splicing forms might affect HCV infection as well. Location of OCLN in a duplicated region complicates genetic mapping. Currently, only a few single nucleotide polymorphisms (SNPs) in the HapMap database (www.hapmap.org/) uniquely map within the full-length OCLN gene. Future studies should explore genetic regulation of OCLN expression in human tissues and whether OCLN isoforms play a role in the clinical outcome of HCV infection.
We thank M. Holz, A. Forest, M. Panis, and A. Webson for laboratory support and C. Murray and Patricia Porter-Gill for critical reading of the manuscript. We thank The Rockefeller University Flow Cytometry Resource Center, supported by the Empire State Stem Cell Fund through NYSDOH contract C023046. The study was supported by the intramural research program of DCEG/NCI/NIH (to L.P.-O. and T.R.O.), PHS grant R01 AI072613, the Greenberg Medical Research Institute, and the Starr Foundation (grant given to C.M.R.).
Opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Fund, the NYSDOH, the State of New York, or the Department of Health and Human Services.
We thank the Liver Tissue Cell Distribution System for liver samples and the Molecular Targets Team, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (DCTD), NCI/NIH, for NCI-60 samples.
Published ahead of print on 12 May 2010.
†Supplemental material for this article may be found at http://jvi.asm.org/.