Expression of individual proteins of several virus species has allowed to elucidate functions of viral gene products. For example, the proteins responsible for triggering the formation of membrane structures involved in viral RNA replication, i.e., the viral replication complex, have been pinpointed in such experiments for poliovirus, hepatitis A virus, and members of the nidoviruses (1
). For HCV, the viral replication complex is not yet characterized. In the present work we show that several structural and nonstructural HCV proteins have the propensity to modify the cellular membrane framework in a characteristic way, some of the alterations being possibly related to the formation of a replication complex. An advantage of protein expression systems is that they presumably do not accumulate adaptive mutations, which are necessary for efficient in vitro propagation of replicons (9
). Thus, the proteins studied are identical to those synthesized during an HCV infection in humans.
The expression of the entire HCV polyprotein induced membrane alterations similar to those found after expression of individual proteins, although there were differences in the extent of membrane transformation. As an exception, the so-called contiguous vesicles were observed only after expression of the entire ORF. Thus, their formation may depend on a concerted action of several viral proteins. The effect of the proteins, however, might be indirect, since by IEM, little if any viral protein was found to be associated with these vesicles. Formally, we cannot rule out that protein NS2, which was not visualized by IEM in the present work, can induce the contiguous vesicles. However, more recently, this type of vesicles was also found in HuH-7 cells harboring a subgenomic replicon lacking NS2 (R. Gosert et al., unpublished data).
The contiguous vesicles have a striking resemblance to the vesicles arising during poliovirus infection. Since the formation of the poliovirus vesicles involves COPII proteins and membrane structures of an altered anterograde membrane pathway (41
), it might well be that the HCV contiguous vesicles also reflect alterations in the exocytotic membrane traffic, brought about by the HCV protein(s).
In cells expressing the NS4B protein alone, the characteristic membrane alteration was a membranous web. In UHCVcon-57.3 cells expressing the entire HCV polyprotein, as well as in liver cells of HCV-infected chimpanzees, this membrane alteration was detected. In UHCVcon-57.3 cells, the web harbored all HCV structural and nonstructural viral proteins tested. Since the actual intracellular site of HCV RNA replication is not yet determined, the significance of the observed membrane alterations induced by HCV proteins cannot at present be assessed for their roles in virus replication. The observations that all HCV proteins associate with the membranous web and that the same structure is found during HCV replication in chimpanzee liver make the web a good candidate for the replication complex. The contiguous vesicles, always found in close proximity to the web, are less likely to represent the replication complex, since there were only few if any viral proteins found to be associated with this structure.
As inferred from the successful construction of subgenomic replicons (34
), the nonstructural proteins NS3 to NS5B are sufficient to induce the putative membrane alterations competent to support viral replication. Since we found only NS3-4A and NS4B, but not NS5A and NS5B, to induce membrane alterations, the NS3-4A-induced vesicles (Fig. and ) and/or the NS4B-induced web (Fig. and ) is likely to be the structure(s) equivalent to the HCV replication complex. At present, we favor the view that both alterations might contribute to the replication complex, since the web carries NS3-4A as well as NS4B protein. In addition, our EM pictures are compatible with an association of NS3-4A- and NS4B-induced structures forming a (higher-order) complex by incorporation of the NS3-4A-induced loose vesicles into a NS4B-induced basic web structure. Such a process would also explain the slight difference in the aspect of the web induced by NS4B alone (Fig. ) compared to the web in cells expressing the entire HCV polyprotein (Fig. ). The two proteins NS5A and NS5B, found not to alter membranes but still to be present on the web, can be posttranslationally targeted to membranes (44
). This might indicate that these two proteins, of which at least the RdRp NS5B is necessary for RNA replication, are attracted and incorporated into a nascent or already completed web to form the final functional replication complex.
Very recently, the NS4B protein of the HCV-related pestiviruses was reported to play a role in cytopathogenicity of the virus (39
). For HCV, no function for NS4B was described so far. Our finding that NS4B induces a distinct membrane structure, possibly related to virus replication, is the first function of HCV NS4B thus far described. Since in other plus-strand RNA viruses, particularly poliovirus, the formation of a replication complex and the induction of cytopathology are coupled (7
), one could speculate that also for HCV, NS4B might be involved in virus-mediated cell injury under certain conditions. Interestingly, adaptive mutations in NS4B have recently been found to greatly enhance replication of HCV replicons in HuH-7 cells (33
At present, it is open whether the membrane structures, particularly the web, observed in the absence of RNA replication will be the same when RNA synthesis occurs. In the poliovirus system, expression of viral gene products in the absence of RNA replication induced structures morphologically indistinguishable from the membrane alterations harboring the viral replication complex found in poliovirus-infected or replicon-transfected cells. Only the location of replicating RNA was different from that of nonreplicating RNA, in that only replicating RNA was associated with the membranous vesicles of the replication complex (20
The mechanism by which mature, infectious HCV progeny is formed is still enigmatic. By analogy to other flaviviruses, formation of virus particles is thought to occur by budding of protein E1- and/or E2-containing membranes, together with core (capsid) protein, into the lumen of the rER (for a review, see references 3
). The E1 and E2 proteins contain ER retention signals (19
), which is compatible with a role of these proteins in the budding process.
In UCp7con-9.1 cells expressing the HCV structural proteins, we were able to visualize the budding process predicted by several authors. The budding was quite extensive, leading to giant rER vacuoles containing large but also very small membrane buds. In UHCVcon-57.3 cells expressing the structural proteins in the context of the entire HCV polyprotein, budding was much less extensive. This may be explained by an overall lower expression level of structural proteins in UHCVcon-57.3 cells than that observed with UCp7con-9.1 cells, and/or retention of the structural proteins in the membranous web, leading to a lower amount of structural proteins available for the budding process. No (empty) virus particles could be found in either UCp7con-9.1 or UHCVcon-57.3 cells. This was expected, since it was found that particle formation requires structured RNA (31
). In addition, it is conceivable that not only virion formation but also release of structural proteins from the web and subsequent migration to the rER has to take place in an RNP complex; thus, it can occur only in a system with ongoing viral RNA replication. In any case, the budding process per se at the rER does not require RNP but can be effected by the structural proteins alone.
Based on the fact that most HCV proteins are membrane-binding proteins, several authors (see reference 3
and references therein) have postulated that at least two replication steps of HCV, i.e., RNA replication and virion formation are (ER) membrane associated. The present paper demonstrates that HCV proteins have the propensity to induce distinct alterations of ER membranes, which may represent, in productively infected cells, the scaffolds necessary for virus multiplication.