Abstract

The hepatitis C virus (HCV) non-structural 4B (NS4B) protein is a 27kDa hydrophobic protein which for many years was characterized mainly as a protein of unknown function. Recently, however, information about the protein and its involvement in mediating various viral activities and effects on host cells is beginning to accumulate. NS4B has been implicated in modulation of NS5B's RNA dependent RNA polymerase activity and various host signal transduction pathways, a possible role in HCV carcinogenesis, impairment of ER function, and regulation of both viral and host translation. Perhaps most significant, NS4B has recently been found to be responsible for the formation of a novel intracellular membrane structure, termed the membranous web, which appears to be the platform upon which viral replication occurs. Specific domains within NS4B have been identified which likely underlie the mechanisms employed by NS4B to mediate many of the preceding functions. As such, these domains which include an amphipathic helix and nucleotide-binding motif represent attractive targets for new antiviral strategies.

Introduction

With the cloning of HCV (Choo et al., 1990) it was possible to deduce many of the expected protein products encoded in the viral genome. Among these was a 261 aa protein now known as NS4B. Unlike several other predicted protein products of the HCV polyprotein, no obvious functions could be immediately ascribed to NS4B. For years, NS4B essentially remained a membrane-associated protein characterized mainly by a lack of known function. Recently, however, considerable information concerning NS4B as begun to accumulate. These efforts have both led to a realization of NS4B's importance to the viral life cycle and yielded attractive new targets for antiviral drug development. This chapter will attempt to review these developments and speculate on some of the future directions in this increasingly exciting field. After discussing NS4B genesis, localization and topology, NS4B's relationship with a specialized type of membrane will be addressed. A survey of various NS4B properties and functions will follow, including NS4B's role in the induction of a novel type of membrane structure which represents the candidate site for HCV replication. Finally, we will focus on features within NS4B which may underlie the mechanisms employed by NS4B to mediate its associated functions.

Proteolytic Generation of NS4B

The HCV genome is translated from a single ~3000 amino acid-long open reading frame into a large polyprotein that is processed both co- and posttranslationally by a combination of host and viral proteases (see Fig 1). Cleavages generating the HCV structural proteins result from the sequential action of the endoplasmic reticulum (ER) resident enzymes signal peptidase (Hijikata et al., 1991) and signal peptide peptidase (Weihofen et al., 2002). Processing of the non-structural protein region is mediated by two virus-encoded proteases: The zinc-stimulated NS2-3 autoprotease cleaves at the NS2/3 junction, and the NS3 serine protease is responsible for liberating the remaining downstream non-structural proteins (Lindenbach and Rice, 2001). Efficient activity of the NS3 protease requires NS4A, a 54 amino acid-long protein which acts as a cofactor for processing at the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites (Bartenschlager et al., 1994; Failla et al., 1994; Tanji et al., 1995). Stable partial cleavage products that include NS4B can also be detected during the cleavage process (see Fig. 1 Bartenschlager et al., 1994). In other positive strand RNA viruses, such partial cleavage products have independent activities, distinct from those associated with the completely processed individual proteins (LaStarza et al., 1994; Parsley et al., 1999). As described further below, there is evidence that at least for one partially-processed precursor including NS4B, HCV may also make use of such a strategy which increases the repertoire of functionally distinct protein-encoded activities. Moreover, although many studies of NS4B examine the effects of NS4B alone, NS4B may also function as part of multiprotein complexes with NS4A and NS5A, with or without NS3 and NS5B (Lin et al., 1997; Neddermann et al., 1999) (Fig. 1).

Fig. 1

Kinetics and possible partial cleavage products of HCV polyprotein processing. The first cleavage event as indicated by the appearance of partial cleavage products is between NS3 and NS4A. NS4A/B cleavage appears to be delayed as shown by the presence (more...)

Subcellular Localization and Topology

Initial studies of NS4B attempted to determine its subcellular localization as a first step towards the understanding of its function. Indirect immunofluorescence and green fluorescent protein (GFP) fusion experiments determined that NS4B is cytoplasmically-localized in the perinuclear region where it adopts chickenwire-like and speckled patterns typical of a membrane-associated protein (Kim et al., 1999; Selby et al., 1993). Later Hugle et al. (Hugle et al., 2001) combined the use of several methods, including specific antibodies and confocal analysis, to show that NS4B was localized to the ER, where it colocalized with the other HCV nonstructural (NS) proteins. This localization has been observed when the protein was expressed either alone or in the context of HCV's other NS proteins, as well as in cells harboring HCV replicons (El-Hage and Luo, 2003; Mottola et al., 2002). Lundin et al. (Lundin et al., 2003) confirmed these localization results and reported that the speckle or foci-like structures not only contained ER markers, but that they tended to be both larger and more common the longer the cells are allowed to express recombinant NS4B.

A recent study examined the localization of NS4B in live cells using a chimeric NS4B-GFP fusion (Gretton et al., 2005). NS4B appeared to be distributed in a thread-like pattern, consistent with ER localization, and at small foci similar to those described by others (Gosert et al., 2003; Moradpour et al., 2004). The authors termed these foci membrane-associated foci (MAFs). The mobility of NS4B in ER membranes and MAFs was assessed using fluorescence recovery after photobleaching (FRAP) experiments. In these experiments fluorescent molecules in a defined area are irreversibly photobleached by a high-power laser. Subsequent diffusion of non-bleached molecules into the bleached area leads to a recovery of fluorescence. Fluorescence intensity in selected regions in live cells expressing the NS4B-GFP protein was measured before and after photobleaching. A topologically related GFP-tagged DNase X was used as a control. NS4B was determined to have reduced mobility in MAFs compared with the ER membrane suggesting that NS4B is likely to form different interactions on MAFs and the ER.

In vitro transcription–translation experiments performed in the presence of microsomal membranes revealed that targeting to the ER membrane is cotranslational. Using classical membrane extraction and proteinase protection assays it was shown that the majority of the protein is cytoplasmically oriented but it was not possible to demonstrate the presence of transmembrane or lumenal fragments (Hugle et al., 2001). This latter finding seemed somewhat puzzling in light of other viral NS4B proteins having at least one transmembrane domain (TMD) and that various computer predictions have estimated that HCV NS4B has several TMDs. The inability to experimentally detect such TMDs could have been for a variety of technical reasons and the lack of available antibodies to facilitate the detection of such fragments.

Another approach to probing NS4B topology involved introducing canonical glycosylation sites at various positions within NS4B. Here the rationale was that the host cell enzymes responsible for glycosylation at such sites are exclusively located within the ER lumen. Thus, those sites contained within NS4B segments which are truly intralumenal would be expected to undergo glycosylation. Only two of the predicted TMDs connected by a putative ER-lumenal loop could be supported experimentally using this method (Lundin et al., 2003). It is possible, however, that the extra amino acids introduced into NS4B in order to insert the target glycosylation sites may have caused unanticipated deleterious changes to NS4B. Another study found that all predicted TMDs could be deleted without impairing NS4B's ability to associate with membranes (Elazar et al., 2004). Within the remaining segments of NS4B, the authors detected a predicted amphipathic alpha helix domain at the protein's N-terminus which was necessary for conferring membrane association upon the mutant NS4B devoid of all TMDs (see more details below). Nevertheless, the authors still favored a predicted NS4B topology containing TMDs, and suggested that the membrane-associating function of the amphipathic helix was more likely to mediate other membrane-associated functions of NS4B beyond simple anchorage to membranes (Elazar et al., 2004). One such function may be to mediate a topologic change of NS4B proposed to occur based upon an unexpected finding of the above-mentioned glycosylation studies. Indeed, Lundin et al. (Lundin et al., 2003) observed that a glycosylation site introduced into the NS4B N-terminal segment—predicted to be cytoplasmically-oriented—was glycosylated in a manner to suggest that it was translocated into the lumen in a fraction of the NS4B molecules. Such cases of alternate topologies have been described in other viruses such as the hepatitis B virus L envelope protein (Bruss et al., 1994) or the M protein from transmissible gastroenteritis corona virus (Escors et al., 2001). The NS4B proteins of the yellow fever and dengue viruses (Cahour et al., 1992; Lin et al., 1993) also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in HCV NS4B. This potential shared topology might support the idea of a common function for NS4B in Flaviviridae (Lundin et al., 2003).

Although the above studies clearly show that NS4B behaves as a membrane-associated protein, the precise nature of this association awaits further definition. A variety of topologies with respect to the ER membrane have been proposed, with NS4B predicted to have between 4–6 TMDs. The N and C termini are expected to be (at least initially) located in the cytoplasm since they are generated by the cytoplasmic NS3 protease (Hijikata et al., 1993; Wolk et al., 2000). Part of the uncertainty stems from the fact that the computer algorithms used to predict these topologies are derived from a databank of solved structures which contain inadequate numbers of membrane proteins.

NS4B and Lipid Rafts

Lipid rafts are cholesterol- and sphingolipid-rich microdomains of cellular membranes which are operationally-defined by their resistance to solubilization with certain non-ionic detergents at 4°C (Cohen et al., 2004; Pike, 2004). Classically, these detergent-resistant membrane microdomains and associated proteins "float" to the low density upper fractions when subjected to density gradient ultracentrifugation—hence the designation of rafts. Lipid rafts are known to play important roles in diverse processes such as signal transduction and protein sorting (Simons and Toomre, 2000; Slimane et al., 2003). Rafts are also exploited by an increasingly recognized number of viruses as portals for viral entry or assembly and release (Campbell et al., 2001; Cuadras and Greenberg, 2003; Manes et al., 2003).

The first indication that HCV might also exploit lipid rafts was the demonstration that both viral proteins and RNA could be detected in low density membrane fractions resistant to solubilization with 1% NP-40 at 4°C (Shi et al., 2003). Although their analysis was limited to NS5A and NS5B, these results suggested that the membranes upon which HCV RNA replication occurs may be lipid rafts recruited from intracellular membranes.

A more detailed analysis revealed that, when expressed together, NS proteins 3 through 5B could all be found in lipid raft fractions (Shi et al., 2003). When expressed individually, however, only NS4B was completely associated with lipid rafts (Gao et al., 2004). Therefore, NS4B, may be the key protein responsible for binding to lipid rafts first and thereby enabling the recruitment or anchoring of other NS proteins in order to form potential replication complexes.

This replication complex-harboring raft may be the same or different from that with which the structural HCV core protein associates (Matto et al., 2004). Moreover, although the raft targeted by NS4B shares biochemical features similar to those of classical plasma membrane rafts, there appears to be some important differences. For one, the steady-state cytoplasmic speckled staining pattern of NS4B is very different from the peripheral surface pattern typical of plasma membrane-based rafts (Matto et al., 2004). Second, a significant amount of the raft-resident HCV NS proteins and RNA are protected from digestion with exogenously added protease and nuclease (Aizaki et al., 2004). This protection is lost upon treatment with raft-solubilizing conditions. Taken together, these data suggest NS4B targets the replication complex components to a specialized raft compartment which is not directly contiguous with the host cell cytosol. It is tempting to speculate that this compartment overlaps with the membranous web (to be described later) and that the vesiculation and physical conformation of the latter helps provide some of the observed protection from experimental nucleases. Similar protection may be provided against host intracellular antiviral mechanisms (Barber, 2001).

The Immunological Effects of NS4B

NS4B is recognized as a target by both the humoral and cellular arms of specific immunity. Historically, NS4B sequences were contained in both the seroreactive clone used to originally isolate the first fragment of the HCV genome (Choo et al., 1989) as well as in the recombinant antigen used in the first generation of anti-HCV commercial assays (Conry-Cantilena, 1997). Indeed some of the most diagnostically relevant antigenic epitopes have been found to reside within NS4B (Chang et al., 1999; Masalova et al., 2002; Rodriguez-Lopez et al., 1999) and this helps explain why NS4B has since been successfully used as an antigenic target in various commercial diagnostic tests for the detection of HCV antibodies in the serum of patients with HCV infection. These highly antigenic properties might prove beneficial for therapeutic purposes as well, such as using NS4B-derived peptides to elicit an antiviral cytotoxic T lymphocytes (CTL) response.

To further characterize some of the mechanisms by which HCV induces an inflammatory and immune response, Kato et al. (Kato et al., 2000) used HCV viral protein–expression vectors cotransfected into mammalian cells with reporter vectors having a luciferase gene driven by various cis-enhancer elements from 5 intracellular signaling pathways associated with cell proliferation, differentiation, and apoptosis. Although core had the strongest effects, NS4B also significantly activated the NF-κB–associated signal. Because the NF-κB pathway is known as an inducer of inflammatory and immune responses, it was therefore suggested that HCV core and NS4B proteins might modulate the production of various cytokines and inflammatory responses in HCV-infected liver.

One of these cytokines appears to be interleukin-8 (IL-8). Indeed, serum IL-8 levels have been shown to be elevated in patients infected with HCV (Polyak et al., 2001b) and IL-8 expression was augmented in Huh-7 cells harboring an HCV subgenomic RNA replicon, compared with the control cells (Kadoya et al., 2005). Expression of NS4B, (and to a lesser extent NS4A) alone were each found to significantly transactivate the IL-8 promoter, resulting in enhanced production of IL-8 protein. The mechanism of IL-8 induction may be via NS4B's effect on NF-κB, as the IL-8 promoter contains a binding site for NF-κB. Because NS5A has also been implicated in the induction of IL-8 (Polyak et al., 2001a), there would appear to be multiple mechanisms whereby such induction can be induced by HCV.

As many cases of HCV are refractory to interferon (IFN) treatment, potential mechanisms underlying HCV resistance to IFN have been the subject of intensive investigation. One approach for studying such HCV resistance to IFN, centered around developing IFN-resistant HCV replicons. For that, cells harboring HCV replicons were subjected to a prolonged low-dose treatment with IFNs. Total RNA derived from these IFN-treated replicon cells was then electroporated into naïve cells and individual cell lines harboring HCV replicons with an IFN-resistant phenotype were isolated. Here too, a possible role of NS4B was found. Indeed, sequencing of the replicons contained in these cell lines revealed that they all shared a single common amino acid substitution in NS4B (Q1737H) which might at least partially explain their IFN-resistant phenotype (Namba et al., 2004). These results should be interpreted with caution since cells cured from the replicon by cyclosporin A, continued to show resistance to IFN suggesting that a host factor(s) rather than replicon RNA(s) could have contributed to the IFN-resistant phenotype. Moreover, direct demonstration that introduction of the Q1737H mutation into a wild type replicon can confer IFN-resistance is still pending.

Modulation of Ns5bs' RNA-Dependent RNA Polymerase Activity

It was recognized early on that HCV NS5B encodes the virus' RNA-dependent RNA polymerase activity required for HCV replication. Many questions about how this activity is controlled, however, remain unanswered.

To study the potential influence of NS3 and NS4B proteins on the priming activity of NS5B, recombinant proteins were generated and introduced into an assay for NS5B's RNA-dependent-RNA-polymerase (RdRp) activity on a template corresponding to the minus strand 3′-untranslated region ((-)3′-UTR) (Piccininni et al., 2002). Physical interactions between NS3 and NS5B as well as between NS3 and NS4B were demonstrated. Both recombinant NS3 and NS4B proteins were also found to modulate NS5B's RdRp activity, but in distinct ways: NS3, via its helicase function, facilitated NS5B activity, whereas this effect was antagonized by the addition of NS4B. These results provide additional evidence that NS4B can function as part of a multi-protein replication complex. Although this data suggests that NS4B might be a negative regulator of NS5B activity in vitro, this need not be the case in vivo where additional regulatory factors may be operative.

Effects of NS4B on Translation

Since many viruses are known to interfere with host translational mechanisms, possible inhibitory effects of HCV proteins on cellular protein synthesis were analyzed using a transient expression system. The core protein, NS4A and NS4B, but not NS3, NS5A or NS5B, inhibited the expression of cell cycle regulator protein p21/Waf1/Cip1/Sdi1 (p21/Waf1) (Florese et al., 2002). There were no significant differences in steady-state p21/Waf1 mRNA levels, as demonstrated by RT-PCR and Northern blot analyses, suggesting the possibility of post-transcriptional inhibition. That the inhibitory effect of NS4B may be at the level of translation was suggested by in vitro translation assays which revealed inhibited synthesis of p21/Waf1 protein when co-translated with NS4B RNA. A similar inhibitory effect of NS4B on the expression of RNaseL was detected although the magnitude appeared to be somewhat smaller. It should be noted that a possible contribution of degradation has not been ruled out.

Using a bicistronic reporter plasmid, the effects of HCV proteins on both cap-mediated (host) and internal ribosome entry site (IRES)-mediated (virus) translation were simultaneously monitored (Kato et al., 2002). In this system, the Renilla luciferase is translated in a cap-dependent manner, while the firefly luciferase is translated from the HCV IRES in a cap-independent manner. Both activities were decreased with the expression of NS4A and/or NS4B proteins suggesting that NS4A and NS4B proteins inhibited both cap-dependent translation and cap-independent translation from HCV IRES. It was suggested that the latter might be a viral self-regulation mechanism limiting the amount of viral protein. In contrast, using a similar bicistronic reporter gene construct, IRES-mediated translation was found to be specifically upregulated in HCV replicon cells (He et al., 2003). No such enhancement was observed when the IRES from either poliovirus or EMCV were substituted for the reporter construct's HCV IRES, suggesting specificity for HCV. Transient expression of individual HCV non-structural proteins in combination with the dual-luciferase reporter construct containing the HCV IRES showed that NS5A and to a lesser extent NS4B could stimulate HCV IRES activity, although the effect was less dramatic than in the context of the entire subgenomic replicon. Reduced phosphorylation levels of both eIF2a and eIF4E were observed in the replicon cells. In the absence of further mechanistic details whereby NS4B may mediate its effects on translation, it is difficult to fully reconcile the above-detailed differences observed by different investigators. Perhaps such discrepancies are due to differences in duration and levels of expression of NS4B, or the presence of a critical host cell factor.

"Membranous Web" Formation

A characteristic feature of plus-strand RNA viruses is their propensity to replicate their genome in close association with host intracellular membranes. These membrane platforms can either be pre-existing membrane organelles or membrane structures induced de novo by the virus (Chu and Westaway, 1992; Froshauer et al., 1988; Lazarus and Barzilai, 1974; Rice, 1996). Egger et al. (Egger et al., 2002) investigated by electron microscopy the capacity of HCV proteins to elicit such intracellular membrane alterations by expressing HCV proteins individually or in the context of the entire HCV polyprotein. Expression of the latter was associated with the induction of a novel membrane structure designated "membranous web" which appeared to consist of vesicles within a membranous matrix. Expression of NS4B alone also induced the membranous web. The emergence of the latter appeared to coincide with a reduction in the rough endoplasmic reticulum (RER), and regions of continuity between the RER and membranous web were observed (Egger et al., 2002). This suggested that the membranous web was derived from the endoplasmic reticulum (ER). Similar structures have been described in livers of HCV-infected chimpanzees (Pfeifer et al., 1980). Immuno-EM experiments revealed that all examined HCV proteins could be found associated with the membranous web, suggesting that these proteins might form a membrane-associated multi-protein complex. Membranous web structures were also found in cells harboring HCV replicons (Gosert et al., 2003). Importantly, viral plus-strand RNA could be localized to these sites using a digoxigenin-labeled riboprobe followed by a gold conjugated anti-digoxigenin antibody. Finally, nascent viral RNA synthesis detected by metabolic labeling with BrU in the presence of actinomycin D (Gosert et al., 2003) co-localized with immunofluorescently-detected NS5A. Similar apparent co-localization of viral RNA and NS proteins has been described by others (Egger et al., 2002; El-Hage and Luo, 2003; Shi et al., 2003). It was therefore postulated that the membranous web represents the candidate site for HCV replication.

It remains to be clarified whether the membranous web, the MAFs described earlier (Gretton et al., 2005), and the speckle-like structures detected by immunofluorescent probes against HCV RNA and proteins in cells harboring subgenomic replicons (Gosert et al., 2003) are all identical structures representing viral replication sites, or different structures with different functions. It will also be important to confirm the existence and character of membranous webs in cells which are capable of permitting all aspects of the viral life cycle (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005).

Nevertheless, NS4B's apparent key role in the establishment of the replication (and possible early assembly) platform represented by the membranous web places NS4B in a particularly prominent position in the viral life cycle. Moreover, because the membranous web is not a normal feature of host cells, selective inhibition of NS4B-induced membranous web formation could represent a specific antiviral strategy with low inherent cytotoxicity.

Modulation of ER Functions

The ER is a major subcellular organelle with which the HCV life cycle is associated. The HCV proteins are translated on, undergo initial processing by, and associate with the ER. Moreover, as mentioned above, the membranous web is postulated to be derived at least in part from the ER. It should therefore not be too surprising that HCV in turn can affect a variety of ER functions.

In poliovirus, two different virally-encoded proteins slow the rate of ER-to-Golgi traffic (Doedens and Kirkegaard, 1995) reducing the rate of ER protein secretion and imparing the presentation of major histocompatibility complex class I (MHC-I) antigens on the cell surface (Deitz et al., 2000). To determine whether HCV proteins might induce similar effects, changes in anterograde traffic from the ER to the Golgi apparatus were determined as a function of HCV NS protein expression (Konan et al., 2003). For this, they monitored the glycosylation status of coexpressed vesicular stomatitis virus G protein (VSV-G), a classical technique for secretory pathway trafficking studies (Lodish et al., 1983). As VSV-G is transported from the ER to the Golgi, the sensitivity of the covalently attached sugars to endo H digestion is altered, thus providing a convenient marker of anterograde trafficking. Of all the NS proteins evaluated, including putative partially-processed precursor proteins, only a fused NS4A/B affected the rate of ER-to-Golgi traffic, reducing it by approximately three fold. Interestingly, no effect was seen with the fully-processed products NS4A or NS4B alone, or in combination. NS4A/B expression inhibited the secretion of other cargo proteins as well. In cells harboring full length HCV replicons, MHC-I appearance on the cell surface was attenuated by three- to five fold compared to control cells. Both NS4A/B and NS4B caused the accumulation of clustered, aggregated membranes in 293T cells and were found localized to these membranes. Only NS4A/B caused the formation of swollen vesicles, but the protein did not localize to these structures. These swollen vesicles were suggested to be ER-derived membranes swollen with cargo due to the blockage in ER-to-Golgi traffic. It was postulated that such blockage–with the associated reduction of cytokine secretion and transport of membrane proteins such as MHC-I to the cell surface--could affect the host immune response to HCV infection.

HCV has also been implicated in the induction of ER stress (for review see Tardif et al., 2005) and this may contribute as well to the decrease in MHC-I expression found in cells harboring HCV replicons (Tardif and Siddiqui, 2003). NS4B may play both a role in the induction of ER stress and its regulation. One mechanism may reside with the ATF6 (activating transcription factor 6) activation associated with HCV replication (Tardif et al., 2002). ATF6 is a transcription factor activated to alleviate ER stress when protein folding is disrupted. Using a yeast two-hybrid assay, cyclic AMP-response-element-binding protein-related protein (CREB-RP), also called ATF6β, was identified to interact with NS4B (Tong et al., 2002). The N-terminal half of NS4B and a central portion of CREB-RP/ATF6β containing the basic leucine zipper (bZIP) domain were involved in this interaction. ATF6α, which shares high sequence similarity with CREB-RP/ATF6β, was also shown to interact with NS4B in yeast although the interaction was weaker than that between NS4B and CREB-RP/ATF6α. Interestingly, ATF6β suppresses transcription of ER stress-inducible genes while ATF6α enhances it (Thuerauf et al., 2004). This might suggest that NS4B can, like NS5A and E2 (Gale et al., 1997; Pavio et al., 2003; Taylor et al., 1999), inhibit specific downstream pathways of ER stress induction. At present, however, interactions of NS4B with ATF6 in vivo and the functional consequences remain to be determined. Finally, it is possible that some of the above effects are consequences of NS4B's interactions with membranes per se, including the diversion of ER components into the creation of the membranous web.

Malignant Transformation

The leading cause of hepatocellular carcinoma (HCC) in the US is hepatitis C virus. Typically, this severe complication of HCV occurs many years after infection, in the setting of cirrhosis. Because the latter is an independent risk factor for HCC, HCV-associated HCC could either be simply an indirect consequence of HCV-induced cirrhosis. Alternatively, the HCC could be the direct result of specific viral factors, presumably in the context of a "multi-hit" scenario where the time course for full accumulation of these hits parallels the development of cirrhosis. In the context of the latter possibility, several HCV proteins including the core protein and non structural proteins NS3 and NS5A have been reported to transform various cell lines, and in the case of core cause tumors when expressed in transgenic mice (Moriya et al., 1998). The cell transformations occur either alone or in cooperation with other known oncogenes (Ghosh et al., 1999; Ray et al., 1996; Ray et al., 2000; Sakamuro et al., 1995). The involvement of HCV's NS protein NS4B in tumor formation was also investigated (Park et al., 2000). NIH3T3 cells co-transfected with NS4B and the Ha-Ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent growth--all characteristics of a transformed phenotype. Similar experiments using c-src, c-fos , c-myc substituted for the Ha-ras, failed to show any tumorigenic phenotypes, suggesting a specificity for enhancement of Ras-mediated pathways. Since many viral proteins are involved in Ras-mediated transcriptional regulation and growth control through AP1 activation, the effect of NS4B on luciferease activity controlled by the AP1 promoter was examined (Park et al., 2000). The luciferase gene was cloned under the control of the AP1 promoter and transfected into NIH3T3 cells stably co-transfected with NS4B and Ha-ras. Luciferase activity in these cells was increased by six fold in comparison with cells stably transfected with Ha-ras alone. AP1-Luc transfection into stable NS4B transfectants did not increase AP1-Luc activity. This suggests that the apparent synergy between NS4B and Ha-ras might be mediated via AP1 activation. Because of the limitations associated with interpreting experiments involving overexpression and in vitro transformation correlates, the relevance of the above (albeit provocative) observations to clinical HCV-associated HCC remains to be determined

NS4B Features That May Underlie the Mechanisms of the Above Functions

The NS4B Amphipathic Helix

Similar to NS5A (Elazar et al., 2003), NS4B has a predicted N-terminal amphipathic helix (see Fig. 2) which suggested another mechanism of membrane association in addition to NS4B's TMDs (Elazar et al., 2004). This amphipathic helix (AH) was found to be conserved across all HCV isolates, suggesting it plays a critical role in productive natural infections. Introduction of mutations designed to disrupt the hydrophobic face of the AH abolished its ability to mediate membrane association.

Fig. 2

The N-terminus of NS4B harbors a predicted amphipathic helix. The amino-terminal segment of NS4B is predicted to adopt an alpha helical secondary structure, depicted here in a helix net diagram wherein the cylindrical alpha-helical segment is "sliced" (more...)

This disruption abolished HCV RNA replication, whereas mutations designed to only partially disrupt the amphipathic nature of the AH resulted in an intermediate level of replication (Elazar et al., 2004). These results genetically validate the NS4B AH as a potential antiviral target, although the mechanistic details underlying the NS4B AH's critical role in replication await further definition. One possibility may be to help mediate the establishment of the HCV replication complex.

When NS4B is expressed from a subgenomic replicon with a mutated NS4B AH, localization of NS4B is aberrant and the cytoplasmic speckle-like pattern typical of wild type replicon cells is lost (Elazar et al., 2004). The mutant NS4B retains a reticular staining pattern suggestive of ER localization, but it is unable to be further sublocalized into the characteristic speckles. Moreover, not only is normal NS4B localization abrogated, but the disrupted NS4B AH prevents other members of the HCV replication complex form coalescing into the speckled pattern associated with replication-competent replicons. Thus the NS4B AH may be responsible for mediating the association of NS4B and replication complex components with lipid rafts. The AH is also hypothesized to play a role in membranous web formation. Interestingly, a second AH has also been identified within NS4B (Glenn and Elazar, unpublished data), which may also play an important role in the viral life cycle.

NS4B has a Nucleotide Binding Motif

Inspection of the NS4B primary sequence revealed the presence of a candidate nucleotide binding motif (NBM) beginning in the middle of the protein. Such NBMs are characterized by conserved of sets amino acids present in proteins known to bind nucleotides. The most conserved elements of NBMs are the so-called A motif (GxxxxGK) and B motif (DxxA) which are separated by a variable number of amino acids, depending on the particular protein (Gorbalenya and Koonin, 1989). Additional motifs common in a large number of GTP-binding proteins, such as the G-protein superfamily, can be identified. Among these are the G and PM2 motifs consisting of single amino acids (F and T, respectively) located between the A and B motifs (Fig. 3).

Fig. 3

Elements of NS4B's nucleotide binding motif (NBM). The NS4B protein is depicted schematically with its 4 predicted TMDs in relation to the ER membrane. The relative positions and amino acid composition of the A motif, B motif, G and PM2 motifs are indicated (more...)

The crystal structures of several G-proteins has revealed that the G and PM2 elements interact with the nucleotide base (guanine in the case of G-proteins) and the chelated Mg⁺⁺ ion, respectively (Stenmark and Olkkonen, 2001).

NS4B was found to specifically bind GTP (Einav et al., 2004). Similar to many other nucleotide-binding proteins, NS4B was also able to hydrolyze nucleotide, indicating it is a GTPase. Mutations disrupting the A motif element of the NBM impaired GTP binding and hydrolysis. These same mutations dramatically inhibited HCV RNA replication, and the effects on GTPase activity paralleled the effect on replication (Einav et al., 2004). Further mutagenesis experiments disrupting the B and the G motifs showed similar effects on viral replication (Moon and Glenn unpublished results). None of these mutations had any apparent effect on NS4B protein levels or its targeting to the ER. Together these results suggest that the nucleotide binding motif within NS4B is essential for mediating NS4B's role in HCV replication in vitro. The requirement of a nucleotide binding motif for productive viral infection in vivo is further suggested by the conservation of this motif across natural HCV isolates of all genotypes. Although it is clear that this NBM mediates critical functions in the viral life cycle the exact details of its function await further definition. One possibility can be that the NS4B NBM mediates binding of nucleotides not only as single molecules but also as part of a polynucleotide structure such as RNA. By simultaneously binding cellular membranes and RNA, NS4B might contribute to the structural integrity of the replication complex by helping to anchor it to membranes.

The ability to bind and hydrolyze GTP has evolved to serve diverse regulatory roles in biology, in part because it represents an efficient and regulateable molecular switch. As such, the NS4B NBM affords a wide variety of potential regulatory mechanisms and it can be readily envisaged to mediate many of the effects ascribed to NS4B in this chapter. Because the amino acids upstream and downstream of the NBM are highly conserved across HCV isolates, yet very different from known host cell G-proteins, there is also the potential for selective inhibition of the NS4B NBM.

Future Directions

As reviewed in the preceding sections, mounting evidence indicates the importance of NS4B to various viral activities. NS4B also appears to be connected with various viral effects on the host cell. It is quite clear that to mediate all these effects NS4B likely has a variety of cellular and/or viral protein partner(s). Uncovering their identity may further clarify some of NS4B's functions—many of which still have unproven mechanisms. Important information might be gained from investigating common features in the NS4B proteins of different viruses from the Flaviviridae family. For example, the related bovine viral diarrhea virus isolates divide into cytopathic and noncytopathic biotypes. In all noncytopathic biotypes that arouse from cytophatic variants an Y2441C substitution in NS4B was found. This might implicate the involvement of NS4B in viral cytopathogenicity (Qu et al., 2001).

Although information about NS4B is continuing to accumulate, several key points relating to its currently ascribed functions remain unclear. For example, only 2 of the four to six predicted TMDs in NS4B have experimental validation. Understanding the exact topology of NS4B could assist in further revealing some of its functions and in the design of specific inhibitors. Another issue awaiting further clarification is the exact intracellular localization of NS4B and its relationship to viral replication. The NS4B-induced membranous webs, MAFs, and the characteristic NS4B speckles may or may not be the same structures. Moreover, which of these represents the authentic sites of viral replication remains to be clarified. NS4B's inhibitory effects on the host translation machinery seem somewhat clear but its exact effect on viral IRES-mediated translation remains uncertain. Improving the understanding of these issues might provide the requisite tools to specifically control viral protein translation. Similarly, the critical role of NS4B's NBM in the viral life cycle has been demonstrated but the exact details of the function(s) mediated by the GTPase activity remain to be fully described. Although there are undoubtedly yet to be discovered features of NS4B, its already identified properties clearly make it a valuable probe of host cell biology.

Both the NS4B AH and NBM provide potential mechanisms to mediate many of the proposed functions for NS4B. The ability to pharmacologically inhibit these domains thus represents another exciting avenue for future research. With respect to the AH, similar strategies as those shown to be effective against the NS5A AH (Elazar et al., 2003) can be readily adapted to the NS4B AH target. The NBM may offer even more readily adaptable antiviral strategies.

Further characterization of the possible role of NS4B in malignant transformation may advance the understanding of HCV-associated carcinogenesis mechanisms and may lead to novel therapeutic strategies. Alternatively, effective pharmacologic eradication of HCV could by itself make the leading cause of hepatocellular carcinoma in the US theoretically preventable. By analogy with other infections, such as tuberculosis or HIV, this type of pharmacotherapy is most likely to consist of a cocktail which includes multiple agents, each designed against an independent virus-specific target. Exploitation of current and yet to be identified targets within NS4B could increase the repertoire of agents available for inclusion in such therapeutic cocktails of the future.

Future Directions

This work was supported by ROIDK066793 and Burroughs Wellcome Career Award (to JSG).

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