The results presented above support a model shown in . CREB3L1 is synthesized as a membrane-bound precursor. In cells infected by a DNA virus such as MHV-68, negative stranded-RNA virus such as Sendai virus, or positive stranded-RNA viruses such as HCV and WNV, CREB3L1 is activated by RIP catalyzed by S1P and S2P. As an analogy to other well-characterized transcription factors activated through RIP, we assume that the viral infection stimulates the translocation of CREB3L1 precursor from the ER to Golgi complex in which S1P and S2P reside (DeBose-Boyd et al., 1999
; Shen et al., 2002
). Following the cleavage, the NH2
-terminal domain of CREB3L1 enters the nucleus, where it activates transcription of genes encoding cell cycle inhibitors to block proliferation of the virus-infected cells. For most of the viruses we analyzed, inhibition of proliferation of their host cells did not affect their replication. Thus, rather than directly inhibiting viral replication, CREB3L1 may play an important role to prevent virus spread by limiting proliferation of virus-infected cells. Among the viruses we studied, HCV is unique in that its replication in Huh7-derived cells requires active division of the host cells (Pietschmann et al., 2001
; Scholle et al., 2004
). As a result, CREB3L1-mediated signaling not only blocks proliferation of virus-infected cells but also inhibits viral replication. However, the in vivo
relevance of the observation remains to be determined. Although hepatocytes do not enter the cell cycle in healthy livers, they proliferate rapidly to repair liver injuries induced by HCV infection. Thus, further studies are required to determine whether CREB3L1 inhibits division of virus-infected hepatocytes in vivo
to limit HCV replication at a low level and to prevent the spread of the virus.
A model illustrating the role of CREB3L1 in limiting HCV infection
It was reported previously that ER stress triggers cleavage of CREB3L1 (Murakami et al., 2006
). ER stress is also known to be induced by massive synthesis of ER-associated viral proteins in cells infected by viruses (He, 2006
). These viruses include those used in the current study such as HCV (Tardif et al., 2002
; von dem Bussche et al., 2010
) and WNV (Medigeshi et al., 2007
)). Thus, it is conceivable that viral infection triggers cleavage of CREB3L1 through ER stress. If this is the case, CREB3L1 may belong to a growing list of proteins that defend against viral infection through ER stress (Martinon and Glimcher, 2010
In the current study, we show that both Huh7.5 and HRP-1 cells are highly permissive for replication of HCV subgenomic replicons derived from the genotype 1 HCV owing to the lack of expression of CREB3L1
. However, only Huh7.5 but not HRP-1 cells can be infected by the JFH1 strain of the genotype 2 HCV. Unlike genotype 1 HCV, genotype 2 HCV is much more sensitive to interferon (Zein, 2000
). This difference between Huh7.5 and HRP-1 cells might be explained by a mutation in RIG-I found in Huh7.5 (Sumpter, Jr. et al., 2005
) but not in HRP-1 cells, which makes Huh7.5 but not HRP-1 cells defective to produce interferon in response to viral infection.
Inasmuch as CREB3L1
inhibits cell proliferation by activating genes encoding proteins that inhibit the cell cycle, it may be considered as a tumor suppressor gene. Indeed, inactivation of CREB3L1
through chromosome fusion is associated with development of low-grade fibromyxoid sarcoma (Mertens et al., 2005
). Considering that CREB3L1 is proteolytically activated in virus-infected cells, the protein may play an important role in preventing virus-induced tumorigenesis. Notably, the ability of CREB3L1 to inhibit cell proliferation has not been observed in previous studies that analyze genes activated by the protein (Kondo et al., 2005
; Murakami et al., 2009
; Vellanki et al., 2010
; Fox et al., 2010
). Unlike the current study, these analyses were not performed in virus-infected cells. We observed that the nuclear form of CREB3L1 activated genes that suppress cell proliferation much more profoundly in Huh7-K2040 cells that harbor an HCV replicon than the naïve Huh7 cells (data not shown). Thus, it is likely that another factor generated in virus-infected cells cooperates with the nuclear form of CREB3L1 to activate these genes.
In addition to genes encoding proteins that inhibit the cell cycle, CREB3L1 also activates transcription of type 1 collagen and genes involved in assembly of collagen matrix as previously reported (Murakami et al., 2009
; Vellanki et al., 2010
). This observation suggests that in addition to preventing proliferation of virus-infected cells, CREB3L1 may also limit the spread of the virus by activating production of small amounts of collagen in virus-infected cells to segregate these cells from their environment. Further study will be required to determine whether chronic deposition of collagen produced in virus-infected cells contributes to fibrosis induced by viral infection.