Like most positive-strand RNA viruses, HCV is expected to replicate its genome on the surface of rearranged intracellular membranes. Ultrastructural evidence for such membranes has been reported in liver biopsy specimens from chimpanzees infected with HCV (18
), in HCV-infected Huh7.5 cells (59
), and in cells expressing either the subgenomic replicon (23
), the entire HCV polyprotein (18
), NS4AB (33
), or NS4B protein alone (18
). Recently, the role of the web in viral-RNA replication has been further strengthened by the localization of nascent RNA on this novel membrane (23
Very little is known about the mechanism of web formation following HCV infection. Using IF, various reports have shown that most of the HCV NS proteins involved in viral RNA replication are associated with the ER membrane (19
), suggesting that the web is derived in part from the ER compartment. In this report, we used IF coupled with immunoisolation of a subcellular fraction containing HCV NS4B to examine the possible contributions of other intracellular compartments in forming the web (18
). We have confirmed that ER markers, including calnexin (42
) (Fig. and ), are associated with the web-inducing protein NS4B. These observations, together with previous reports (19
), clearly suggest that the web is derived in part from the ER compartment.
It is not clear whether the web is derived from a single intracellular compartment, from a single compartment incorporating markers from other intracellular membranes, or from a unique pooled compartment. For instance, the poliovirus-induced double membrane vesicles have been reported to derive from the ER by a mechanism that selectively excludes ER-resident proteins (67
); interestingly, these membrane vesicles also appear to contain markers specific for the Golgi and the lysosomal compartments (61
). Kunjin virus, a flavivirus closely related to HCV, induces membranes derived in part from the ER and the trans
-Golgi network (70
). We did not find that the web contains markers associated with the Golgi, lysosomal, or mitochondrial membranes (data not shown), nor was Golgi-associated protein, golgin 97 (35
), immunoisolated with HCV NS4B protein (Fig. ). These results suggest that the web is not derived in part from these compartments, but we cannot rule out the possibility that a marker from one of these membranes is selectively incorporated into the web.
Using IF and immunoisolation approaches, we found that Rab5, an EE protein, is associated with the web-inducing protein NS4B (Fig. and ). To determine the extent of involvement of the EE membrane in web formation, we examined colocalization between NS4B and Rab5 effectors, including EEA1 (63
) and rabaptin 5 (27
). Syntaxin 13 (10
), an EE integral membrane protein, and Rab4 (39
), a protein transiently associated with the EE, were also examined for their association with NS4B. All these markers, especially those tested (EEA1 and Rab4), were found to costain with endogenous Rab5B protein under IF conditions (data not shown). Interestingly, as reported by Lundin et al. (42
), we have also found that NS4B does not always colocalize with EEA1 when expressed alone (data not shown). However, EEA1, rabaptin 5, and Rab4 were found to be associated with NS4B in the context of the full-length and subgenomic NS5A-GFP replicons. Furthermore, like Rab5, most of these EE proteins were immunoisolated, along with NS4B (Fig. ). These results are consistent with our current hypothesis stating that the web is formed in part from the ER compartment through selective retention of a factor(s) from other intracellular membranes or from a unique pooled compartment including the ER and other intracellular compartments. However, it is also possible that the web actually consists of a heterogeneous population of membranes. The current results do not allow us to distinguish between these possibilities. Further biochemical characterization of the immunoisolated fraction, coupled with IF and electron microscopy, will help to determine the origin of the web.
It is intriguing that Rab5 or any other EE marker is an intrinsic component of the web. Rab5 belongs to the Ras superfamily of small GTPases; it is involved in the regulation of membrane fusion in the early endocytic pathway (11
). Rab5 has three isoforms, Rab5A, -B, and -C (11
). These isoforms localize to the EE, regulate endocytosis (11
), and share close to 85% amino acid sequence identity. Rab5 activity has been linked to the H-Ras signal transduction pathway, and expression of oncogenic H-Ras is known to stimulate endocytosis (5
). Activation of the H-Ras signal transduction pathway, including phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt, appears to be directly linked to Rab5 activation and endocytosis, since DN Rab5 blocks endocytosis by the H-Ras pathway (4
). Interestingly, activation of the N-Ras-PI3K-Akt pathway has been reported in Huh7 cells expressing HCV replicon (46
). These findings, coupled with the report that PI3K interacts directly with both Ras and Rab5 (58
), suggest that HCV might target the signal transduction pathway between Ras and Rab5 activation of endocytosis. Several pathogens are known to target Rab5 function. For example, phagosomes containing Mycobacterium tuberculosis
are reported to bind tightly to Rab5, leading to delayed phagolysosome maturation (54
). The SpoE protein of Salmonella
acts as a Rab5-specific nucleotide exchange factor; it recruits nonprenylated Rab5 on Salmonella
-containing phagosomes to promote fusion with EE and prevents phagolysosome maturation (51
). Aside from the role of Rab5 in adenovirus cell entry (57
), there is no known report of direct involvement of Rab5 in viral-genome replication. Rotavirus VP4 spike protein, for example, has been reported to interact with Rab5 protein (20
), but the role of Rab5 in the virus life cycle has not been elucidated yet. Replication of the coronavirus mouse hepatitis virus has been reported to occur on late endosomal membranes (68
), but not on the EE. Thus, HCV appears to be the first reported virus whose replication may require EE-derived membranes.
We tested the hypothesis that Rab5 plays a role in the formation of the web and perhaps in HCV genome replication by using siRNAs specific to Rab5 isoforms. We found that endogenous Rab5 can be partially silenced without any noticeable effect on cell viability (data not shown); this silencing resulted in a decrease in viral RNA synthesis. Using labeled Rab5 siRNAs, we examined the relationship between viral-RNA synthesis and web formation. The presence of labeled Rab5 siRNAs resulted in a substantial decrease in Rab5B and GFP fluorescence from the subgenomic NS5A-GFP replicon. In addition, using a WT, CA, or DN form of Rab5A, we have found that DN Rab5A expression results in a drastic decrease in GFP fluorescence from the subgenomic NS5A-GFP replicon. These results suggest that Rab5 may play a direct role in web formation. However, we cannot rule out the possibility that a decrease in GFP fluorescence is the result of a decrease in HCV RNA synthesis, nor can we rule out the possibility that the effect of Rab5 silencing and DN Rab5A on HCV synthesis could be indirect, due to interference with the formation of EEs.
In an attempt to determine how EE proteins are retained in the web, we examined possible interaction between Rab5 (the first EE protein shown to colocalize with NS4B) and HCV NS4B protein. Using a co-IP assay, we have shown that Rab5A interacts with HCV NS4B when expressed alone (Fig. ), but not in the context of the full-length replicon (data not shown). We did not observe interaction between NS4B and Rab4 or NS4B and EEA1 (data not shown). However, by combining subcellular fractionation with NS4B immunoisolation, we have shown that a purified fraction containing HCV replicase proteins, including NS4B, also contains the EE proteins Rab5, syntaxin 13, and Rab4. Taken together, these results suggest that Rab5 is retained in the web through its direct interaction with NS4B or indirectly through its association with the EE. Studies are under way to examine in more detail the significance of Rab5 presence in the web and in the HCV life cycle.