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
). 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. ). 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.