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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Chapter 9HCV NS5A: A Multifunctional Regulator of Cellular Pathways and Virus Replication

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The hepatitis C virus (HCV) non-structural 5A (NS5A) protein has generated wide interest in HCV research because of its ability to modulate the host cell interferon (IFN) response. The protein is phosphorylated on multiple sites by host cell kinases and interacts with host cell membranes. While no known enzymatic function has been ascribed to NS5A, it is an essential component of the HCV replicase and exerts a wide range of effects on cellular pathways and processes, including innate immunity and host cell growth and proliferation. In this chapter, we review the many studies describing the interaction of NS5A with viral and host cell proteins, its ability to modulate multiple cellular pathways, and its recently described structural attributes, subcellular localization, and function during HCV replication.


Translation of the HCV genome results in the production of a large polyprotein, from which NS5A is processed by the NS3 protease (Reed and Rice, 2000). As a nonstructural (NS) protein with no apparent enzymatic activity, NS5A functions through interaction with other viral and cellular proteins. Its primary amino acid (a.a.) sequence predicts a proline-rich, predominantly hydrophilic protein with no obvious trans-membrane helices. NS5A exists as multiple phospho-isoforms and is predominantly localized in the cytoplasmic/perinuclear compartments of the cell, including the ER and the Golgi apparatus. This pattern of NS5A localization is consistent with the notion that NS5A interacts with multiple host cell and viral proteins. There is strong evidence that NS5A is also localized in certain modified cytoplasmic membrane structures during HCV replication, where it plays a functionally significant role as part of the HCV replication complex or replicase. NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions, and viral pathogenesis.

Structural Features and Subcellular Localization of NS5A

Early studies utilizing cells in which NS5A had been overexpressed or liver biopsy samples from chronic HCV patients, showed that NS5A is localized in the cytoplasm and the perinuclear membrane fraction, consistent with localization to the ER/Golgi (Ide et al., 1996; Polyak et al., 1999; Tanji et al., 1995a). In addition, when expressed either alone or in the context of additional HCV NS proteins (an NS3-5B polyprotein) in human hepatoma cells, NS5A was also found to co-localize with the HCV core protein on the surface of globular structures containing lipid droplets (Shi et al., 2002). Interestingly, previous studies had noted that NS5A binds to the core protein on membrane structures (Goh et al., 2001). Also, NS5A was found to bind to Apolipoprotein A1 (ApoA1), a protein component of high-density lipoprotein (HDL) particles and co-localized with ApoA1 in the Golgi (Shi et al., 2002). In other studies, NS5A was found to bind to a snare-like protein called hVAP-33 (Tu et al., 1999). In HCV replicon cells, NS5A bound to hVAP-33 and localized to detergent-resistant lipid rafts (Gao et al., 2004). In a study utilizing an HCV replicon in which NS5A was fused to green fluorescent protein (GFP), the NS5A-GFP fusion protein was associated with brightly fluorescent dot-like structures in the cytoplasm (Moradpour et al., 2004). Analysis of these structures by electron microscopy led to their description as "membranous webs". It was suggested that these might represent sites of bound replication complexes or sites of virus assembly since HCV NS proteins and nascent viral RNA all co-localized to these structures (Moradpour et al., 2004). Given its interaction with host cell proteins and membranes, as well as the HCV core protein (Goh et al., 2001), it is possible that NS5A may also regulate HCV virus assembly directly through its interaction with the viral capsid protein. So it seems that after its expression and processing in the ER, the NS5A protein localizes into specialized cytoplasmic membrane structures, as a part of putative HCV replication and/or assembly complexes. These specialized membrane structures may be derived from or related to either ER or Golgi, and the association of NS5A with these structures may require interaction with host-derived, membrane-associated proteins or other viral proteins. The importance of cellular membranes is underscored by the recent finding that inhibitors of protein geranylgeranylation (Ye et al., 2003) and fatty acid biosynthesis (Kapadia, 2005) block HCV replication in replicon cells. Recently, FBL-2, a geranylgeranylated cellular protein was shown to bind to NS5A and found to be critical for HCV replication (Wang et al., 2005). Whether or not additional cellular proteins play a role in these processes is not known.

The membrane association of NS5A protein occurs post-translationally and NS5A has similar properties to that of an integral membrane protein (Brass et al., 2002). A membrane-anchoring region was mapped to the N-terminal 30 a.a. of NS5A which form a highly conserved amphipathic α-helix (Brass et al., 2002). It was suggested that membrane anchorage is mediated by the hydrophobic side of the amphipathic helix, resulting in an orientation parallel to the lipid bilayer, while positioning the helix in the cytoplasmic leaflet of the ER membrane. A follow-up study demonstrated that the N-terminal amphipathic helix is not only necessary and sufficient for membrane localization, but also important for HCV replication since mutations disrupting helix formation impaired HCV replication (Elazar et al., 2003). A three-dimensional structure of this region was solved by NMR spectroscopy (Penin et al., 2004). The structure revealed an α-helix extending from a.a. 5 to 25. The helix contains a hydrophobic side embedded in detergent micelles, and a solvent-exposed, polar, charged side. Confirmatory studies showed that the NS5A membrane anchor region forms an in-plate, amphipathic α-helix, embedded in the cytosolic leaflet of the membrane bilayer. It was also suggested that this region is not only involved in membrane localization, but also required for additional functions, as mutations of conserved residues on the cytosolic face impaired HCV replicon RNA replication without affecting membrane association (Penin et al., 2004). In addition to its role in localizing NS5A protein to appropriate membrane compartments, it is likely that the membrane anchor region provides a platform for protein-protein interactions involved in the HCV replication process. It is also possible that specific cytosolic residues of the helix may contribute to protein-protein interactions with additional viral and cellular components.

Until recently, structural information on the NS5A protein had been limited, largely due to the difficulty in purifying the full-length protein. Early studies on NS5A structure were limited to individual structural motifs and their functions. With the recent characterization of its domain organization and resolution of a structure of the N-terminal region (Tellinghuisen et al., 2004; Tellinghuisen et al., 2005), we can begin to gain an appreciation for the multi-dimensional structure of the NS5A protein. A recent study using bioinformatics-assisted modeling suggested a three-domain organization (Tellinghuisen et al., 2004) with domain I (a.a. 1–213) located in the N-terminal region, and Domain II (a.a. 250–342) and Domain III (a.a. 356–447) in the C-terminal region (Fig. 1). This organization was confirmed by limited proteolysis experiments. Interestingly, an unconventional zinc-binding motif was predicted to exist in the N-terminal domain, indicating that NS5A is a zinc metalloprotein (Tellinghuisen et al., 2004). The predicted zinc-binding motif involves four cysteine residues (C39, C57, C59, and C80; Fig. 1), and includes a structural motif (CX17CXCX20C) that is well conserved among Hepaciviruses and Pestiviruses. In this same study, the zinc content of purified NS5A protein or the N-terminal domain alone was determined and it was found that each protein molecule coordinates one zinc atom. This motif appeared critical for the structural stability and function of the NS5A protein, since mutation of any single cysteine residue in the motif disrupted the ability of NS5A to coordinate zinc and eliminated HCV replicon RNA replication (Tellinghuisen et al., 2004). A more recent study reported the crystal structure of NS5A Domain I (a.a. 36–198) at 2.5-A resolution (Tellinghuisen et al., 2005). The structure revealed the presence of a novel fold, a zinc-coordination motif, and a C-terminal disulfide bond. Mutational analysis suggested that the disulfide bond is not required for the HCV replicase functions of NS5A (Tellinghuisen et al., 2004). These studies have provided a nice starting point for understanding the structural organization of NS5A, the elucidation of structural assembly points of NS5A as it pertains to its role as an HCV replicase subunit, and NS5A's ability to interact with multiple host cell proteins and molecules.

Fig. 1. Schematic diagram of the NS5A protein.

Fig. 1

Schematic diagram of the NS5A protein. Several prominent features of the NS5A protein and described in detail in this review are shown. The three domain structure of NS5A (Tellinghuisen et al., 2005) is depicted. The N-terminal amphipathic α-helix (more...)

While most studies have focused on the membrane associated forms of NS5A, an early study identified a putative nuclear localization signal (NLS) sequence (PPRKKRTVV; a.a. 354–362) within the C-terminal half of NS5A (Ide et al., 1996) (Fig. 1). This sequence appeared to function as an NLS since it was able to target a heterologous protein (β-Galactosidase of E. coli) into the nucleus. The presence of an NLS suggests a possible nuclear localization and function of NS5A in addition to its membrane bound isoforms. One study suggested that the localization of NS5A to membranes is at least partially determined by its most N- terminal region (Satoh et al., 2000). It was found that NS5A mutants lacking this region were localized in the nucleus. Conversely, the N-terminal 27 a.a. from NS5A were capable of retaining a nuclear protein in the cytoplasm. In addition, a cleaved form of NS5A protein missing the N-terminal region (a.a. 155–389) also localized to the nucleus. The N-terminal sequence was able to block the function of the NLS in the C-terminal region and prevented NS5A protein from being transported into the nucleus (Song et al., 2000). This putative "NLS-masking-sequence" in the N-terminus, which appears to overlap with the amphipathic α-helical region, did not function as a nuclear export signal. So it seems that the this region can also regulate the function of the NLS and thus the nuclear localization of NS5A protein, presumably by preferentially targeting NS5A protein into cytoplasmic membrane structures. It is likely that the localization of NS5A protein in different subcellular compartments is determined and regulated by different structural features and/or different forms of the protein, and the differential localization of NS5A in different compartments may contribute to its different biological functions. In particular, the cytoplasmic vs. nuclear localization and function of NS5A could be carefully counter-regulated and balanced through its different structural motifs regulating subcellular localization of the protein during the viral life cycle. Along these lines, the C-terminal half of NS5A contains a positively charged region enriched with acidic and proline residues, a structural feature resembling those of eukaryotic transcriptional activators (Chung et al., 1997; Ide et al., 1996). Following deletion of the N-terminal membrane anchoring domain, the C-terminal half of NS5A functioned as a potent transcriptional activator when fused to the DNA-binding domain of yeast GAL4 protein, in both yeast and human hepatoma cells (Chung et al., 1997; Kato et al., 1997; Tanimoto et al., 1997). Furthermore, a region between a.a. 130–352 was found to be critical for optimal transcriptional activation (Tanimoto et al., 1997). These studies suggest that truncated forms of NS5A may localize to the nucleus via the cryptic NLS only after removal of the N-terminal membrane-anchoring region and regulate cellular gene transcription. The mechanism of NS5A nuclear localization may involve proteolytic processing of NS5A. Indeed, this was observed and a cleaved form of the protein was able to localize to the nucleus and caused transcriptional activation when the alpha subunit of PKA was co-expressed (Satoh et al., 2000; Song et al., 2000). The NS5A cleavage in mammalian cells was enhanced by apoptotic stimuli and was inhibited by the caspase inhibitor Z-VAD-FMK, suggesting that a caspase-like protease(s) contributes to the cleavage of NS5A (Satoh et al., 2000). A later study showed that NS5A protein was also cleaved following induction of apoptosis by the HCV core protein and that the proteolytic processing of NS5A could be inhibited by Z-VAD-FMK (Goh et al., 2001). These studies indicated that NS5A protein cleavage is likely mediated by caspase(s) and/or related protease(s) and may be linked to the induction of apoptosis. In support of this, a recent study found that NS5A was processed into multiple forms in different mammalian cell types (Vero, HepG2, Huh-7, and WRL68), and suggested that both caspase-like proteases and calcium-dependent calpain proteases were involved in NS5A processing (Kalamvoki and Mavromara, 2004). However, this study also showed that both the cleaved and full-length forms of NS5A exhibited a cytoplasmic/perinuclear localization. Although these results suggest that additional proteolytic processing of NS5A may occur, the biological function the cleaved forms in the context of HCV biology remain uncertain at the moment.

NS5A Phosphorylation: A Functional Role or Red Herring?

Studies on NS5A expressed in tissue culture revealed predominantly two forms of NS5A protein with differing apparent molecular weights of 56 and 58 kDa. Basal phosphorylation results in expression of the 56 kDa isoform while hyperphosphorylation results in the 58 kDa form(Kaneko et al., 1994; Tanji et al., 1995b). In addition, there is evidence that p58 is converted from p56 and requires polyprotein processing (Neddermann et al., 1999). Phosphorylation of NS5A protein occurs predominantly on serine residues, with a minor fraction on threonine residues (Kaneko et al., 1994; Reed et al., 1997; Tanji et al., 1995b). A number of serine residues (2194, 2197, 2201, and/or 2204) in the central region of NS5A were found to be important for hyper-phosphorylation, and two other regions (a.a. 2200–2250 and the C-terminal region) appeared important for basal-phosphorylation (Tanji et al., 1995b) (Fig. 1). In addition, a major phosphorylation site was identified as serine 2321, which is located within the C-terminal Class II proline-motifs and likely represents a basal phosphorylation site (Reed and Rice, 1999). In another study, Katze and colleagues identified the major phosphorylated residue on an NS5A phosphopeptide (a.a. 2193–2212) as serine 2194, which is well conserved among HCV genotypes and presumably is a site for hyper-phosphorylation (Katze et al., 2000). Additional phosphorylation sites remain to be mapped. In addition, phosphorylation of NS5A on tyrosine has not been reported. Interestingly, NS5A proteins from other viruses closely related to HCV, such as BVDV and YFV, were also phosphorylated in various in vitro and in vivo systems, and it appeared that phosphorylation occurred via serine/threonine kinase(s) (Reed et al., 1998). These results indicate that NS5A phosphorylation is a well-conserved feature, and either the phosphorylation of NS5A itself or NS5A interaction with its cellular kinases plays an important role in the Flavivirus life cycle. Whether these NS5A proteins from the different virus species are phosphorylated by the same or related kinase(s) is completely unknown. However, one study reported that NS5A protein from HCV genotype-2a was not hyperphosphorylated in contrast to that of NS5A from genotype-1 (Hirota et al., 1999). This raises the question as to whether the same phosphorylation pattern is prevalent throughout all HCV genotypes/isolates, and whether different phosphorylated forms of the NS5A protein play different roles in viral pathogenesis or in the HCV viral life cycle. It is also possible that the different NS5A phosphorylation patterns in vitro were caused by differences in the experimental systems employed. Unfortunately, due to technical limitations, the phosphorylation of NS5A in the liver of chronic HCV patients is not easily addressed.

Numerous studies have attempted to identify the cellular kinase(s) responsible for NS5A phosphorylation. NS5A protein was found to stably associate with an unknown protein kinase(s) from mammalian cells, and this kinase was able to phosphorylate native NS5A protein on serine residues in vitro (Ide et al., 1997). This same study also showed that the catalytic subunit of cAMP-dependent protein kinase A (PKA) was capable of phosphorylating NS5A in vitro. Interestingly, as previously noted, co-expression of the alpha subunit of PKA seemed to affect the transcriptional activity of a cleaved form of NS5A (Satoh et al., 2000). However, there is no evidence that PKA is an NS5A kinase in mammalian cells. By testing the effect of various kinase inhibitors on NS5A phosphorylation in vitro and examining the context of known phosphorylation sites, other studies suggested that the NS5A kinase(s) belongs to the CMGC group of serine-threonine kinases and is likely a proline-directed kinase (Katze et al., 2000; Reed and Rice, 1999; Reed et al., 1997). Indeed, casein kinase II (CK II), a member of the CMGC kinase family, was found to phosphorylate NS5A protein in vitro, and it showed the same molecular size and properties as an unknown kinase that stably associates with NS5A in mammalian cells through the N-terminal region of NS5A (Kim et al., 1999). Thus, CK II stands as a candidate kinase for NS5A phosphorylation, but direct evidence for its role in vivo remains elusive. A more systematic approach was employed by Coito and colleagues, who performed a global screening of all yeast kinases capable of phosphorylating NS5A in vitro, and then attempted to predict and identify homologous mammalian kinases that were also capable of phosphorylating NS5A through both bioinformatic and biochemical methods (Coito et al., 2004). By comparing in vivo and in vitro NS5A phosphopeptide profiles, their results suggested that several mammalian kinases (AKT, p70S6K, MEK1, and MKK6) might be responsible for NS5A phosphorylation in vivo. In particular, the functional relevance of p70S6K or related kinases was further supported by the fact that rapamycin was able to reduce the phosphorylation of specific NS5A phosphopeptides in vivo. Given the complexity of NS5A phosphorylation, it is likely that multiple kinases are involved and that phosphorylation occurs in a regulated and coordinated manner.

Several lines of evidence suggest that the pattern NS5A phosphorylation is dependent on additional HCV NS proteins. One group reported that the level of the hyperphosphorylated form of NS5A (p58), was enhanced by the presence of NS4A. Additionally, the association of NS5A with NS4A through a.a. 2135–2139 of NS5A was important for NS4A-dependent phosphorylation (Asabe et al., 1997; Kaneko et al., 1994). A later study suggested that the appearance of p58 required NS2 in cis and the autoproteolytic activity of the NS2-3 protease. The loss of p58 by disruption of NS2-3 autoproteolysis was rescued by expressing an NS2-3 in trans (Liu et al., 1999). However, other studies published at the same time showed that the presence of NS3-4A-4B in cis was necessary and sufficient for the hyperphosphorylation of NS5A (Koch and Bartenschlager, 1999; Neddermann et al., 1999) and the presence of NS3-4A protease activity in cis was absolutely required for p58 production (Neddermann et al., 1999). Interestingly, it was also found that single a.a. mutations with NS3, as well as mutations within NS4A and NS4B that do not disrupt polyprotein processing, also affected NS5A hyperphosphorylation (Koch and Bartenschlager, 1999). In summary, the exact requirement for other HCV NS proteins and their roles in NS5A phosphorylation is not completely understood, but it seems likely that NS5A phosphorylation is regulated in the context of other NS proteins, and requires both polyprotein processing and interactions among the NS proteins within a multi-subunit protein complex. Despite these observations, only recently has the role of NS5A phosphorylation been described in the context of HCV replication (Evans et al., 2004). These latter studies suggest that the differential phosphorylation of NS5A regulates its function during HCV replication, presumably by affecting its interaction and formation of protein complexes with other proteins.

Emerging Role of NS5A in HCV Replication

Studies utilizing subgenomic HCV replicons in cell culture systems suggest that NS5A plays an important role in the establishment of high-level HCV RNA replication (see Chapter 11). Several adaptive mutations that confer higher replication efficiency to HCV replicons are clustered in the NS5A region, and some of these adaptive mutation sites either overlap with putative NS5A phosphorylation sites or have been shown to affect NS5A hyperphosphorylation. This re-opened the question as to whether NS5A phosphorylation plays a role in HCV replication. Interestingly, when expressed alone in mammalian cells in culture, NS5A has an apparent half-life of four to six hours (Polyak et al., 1999). In replicon cells, the hyperphosphorylated (p58) form of NS5A is much less stable than the basally phosphorylated (p56) form of the protein (Pietschmann et al., 2001), suggesting possible differences in function. Indeed, one study found an inverse relationship between NS5A phosphorylation level and its interaction with hVAP-33, which in turn, is required for HCV replication in the replicon system (Evans et al., 2004). In addition, some of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and increased NS5A binding to hVAP-33. It is noteworthy that the region of NS5A that interacts with hVAP-33 encompasses the putative NS5A hyperphosphorylation sites (Fig. 1). NS5A also binds directly to the NS5B viral polymerase both in vitro and in vivo (Shirota et al., 2002). This interaction was suggested to modulate the enzymatic activity of NS5B (Shirota et al., 2002) and in replicon cells shown to be critical for HCV replication (Shimakami et al., 2004). A model has been proposed in which the phosphorylation status of NS5A serves as a molecular switch in the regulatory process of HCV RNA replication by affecting the association between NS5A and other components of the viral replication complex (Evans et al., 2004). This model also implies that host cell kinases regulate the HCV replication process through differential NS5A phosphorylation. In line with this working model, another study identified three undisclosed kinase inhibitors that blocked NS5A hyperphosphorylation in cell culture, and showed that treatment with any of these compounds stimulated replication of a wild-type replicon construct that has no adaptive mutations and replicates poorly otherwise (Neddermann et al., 2004). This is an exciting finding since this approach might allow efficient replication of many HCV strains that otherwise replicate very poorly in cell culture. This method may also open a way to establish different HCV replicon strains without the introduction of adaptive mutations. Thus, identifying the physiologically relevant NS5A kinases (and phosphatases) remains a high priority. Another prediction from the above working model is that p58, the hyperphosphorylated form of NS5A, is specifically linked to down-regulation of HCV RNA replication in cell culture. This prediction is further supported by results from a recent study, in which extensive mutagenesis analysis was carried out on a region of NS5A presumably involved in basal- and hyperphosphorylation (Appel et al., 2005). It was found that mutations in the central serine cluster reduced NS5A hyperphosphorylation and increased HCV replication. On the other hand, mutations of the C-terminal serine residues decreased the formation of p56, but did not affect HCV RNA replication significantly. Another study showed that the expression of a wild-type NS5A protein, or the introduction of a wild-type NS5A replicon in trans inhibited replication of NS5A-adapted replicons, in a dominant-negative fashion (Graziani and Paonessa, 2004). These results indicate that hyperphosphorylated wild-type NS5A may compete with the adapted-NS5A protein and down-regulate HCV RNA replication.

Despite these exciting results from HCV replicon-based studies, it has been shown that the adaptive mutations, especially those negatively affecting NS5A hyperphosphorylation, inhibit HCV replication following infection of chimpanzees (Bukh et al., 2002). In addition, similar adaptive mutations have not been observed in HCV patients. In fact, the putative hyperphosphorylation sites of NS5A are well conserved among different HCV genotypes/isolates from patients. These observations raised the concern over the physiological relevance of adaptive mutations in the HCV replicon system. In addition, questions as to whether the hyper-phosphorylation of NS5A serves additional biological roles during HCV infection in vivo have arisen. We may speculate that the hyper-phosphorylation of NS5A serves as a switch point between HCV RNA replication and downstream steps, such as virus capsid/particle assembly or virus particle maturation and release and that the hyperphosphorylated form of NS5A may be actively involved in these downstream events. As previously mentioned, an interaction between NS5A and the core protein has been noted (Goh et al., 2001; Shi et al., 2002). This model suggests that the basal- and hyper-phosphorylation of NS5A are regulated in a temporal fashion, presumably by different cellular kinases at different steps of the viral life cycle, to facilitate a complete, productive infection in vivo. With the recently establishment of bona fide HCV infection system in cell culture (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005) (see Chapter 16), now it is possible to examine the differential phosphorylation status of NS5A and its role during different steps of the HCV infection cycle. Additional modes by which NS5A might affect HCV replication have also been suggested. Most recently, NS5A was shown to bind with high affinity to the 3′ ends of HCV plus- and minus-strand RNAs (Huang et al., 2005). NS5A might also indirectly regulate HCV replication by modulating HCV IRES-dependent translation (He et al., 2003; Kalliampakou et al., 2005; Wang et al., 2003) and its ability to modulate cellular antiviral pathways stimulated by IFNs has been well documented (Gale and Foy, 2005; Tan and Katze, 2001).

NS5A as a Viral Interceptor of Cellular Pathways

Interaction with and modulation of host cell signaling pathways constitute an important aspect of many viral life cycles. HCV is no exception to this, and NS5A in particular may play a pivotal role in the interaction between HCV and cellular signal transduction pathways. The interplay between NS5A and the IFN system as well as the role of NS5A in IFN resistance has generated intense interest and has been extensively studied (Gale and Foy, 2005; Tan and Katze, 2001). In this section, we will focus on the affects of NS5A on additional cellular signaling pathways including those involved in growth, cell-cycle control, apoptosis and cell survival, and cellular stress responses.

NS5A has been shown to interact with a wide variety of host cell proteins and thus may modulate numerous diverse signal transduction pathways (Table 1). Among the cellular signaling pathways affected by the NS5A protein, the best characterized are those relating to cell proliferation and cell-cycle control, apoptosis and cell survival, and cellular stress responses. Despite many interesting results and insightful working models, in only a few cases has the functional relevance in the context of HCV replication been addressed. Thus, in most cases, the observations discussed below need further verification in model systems that can better simulate the HCV life cycle in vivo.

Table 1. Proteins reported to interact/associate with NS5A.

Table 1

Proteins reported to interact/associate with NS5A.

NS5A contains proline-rich PXXP motifs representing binding sites for SH3-domain containing proteins (Fig. 1). These motifs are frequently present in cellular signaling molecules (Tan et al., 1999). By testing a panel of SH3 domain-containing cellular proteins, Tan and colleagues found that NS5A specifically interacted with Grb2, a cellular adaptor protein involved in the growth factor signaling. The interaction of Grb2 with NS5A occurred through the C-terminal PXXP motif of NS5A (He et al., 2002; Tan et al., 1999). This interaction seemed to be mediated by the two SH3 domains of Grb2 in a cooperative fashion. Consistent with these findings, EGF stimulation of cells expressing NS5A showed reduced ERK and p38 MAPK activation, which are downstream signaling events mediated by the Grb2 adaptor protein. In addition, NS5A containing mutations within the C-terminal proline-rich motif neither interacted with Grb2, nor blocked EGF-stimulated ERK phosphorylation, supporting the direct connection between NS5A interaction with Grb2 and its effect on downstream MAPK pathways. The NS5A-Grb2 interaction and the inhibition of ERK phosphorylation by NS5A were also shown by another group in various mammalian cell types infected with recombinant HSV-1 viruses carrying NS5A (Georgopoulou et al., 2003). These studies suggest that NS5A can disrupt the MAPK mitogenic pathway through direct interaction with Grb2, either by preventing the recruitment of Grb2 to the upstream receptor complexes, or by disrupting Grb2 interaction with downstream components of the pathway, such as Sos. However, the original study found no evidence that NS5A reduced Grb2-Sos association. More recent studies have found that in HCV replicon cells there was reduced EGF receptor tyrosine phosphorylation and aberrant recruitment of the Shc and Grb2 adaptor proteins to the receptor. This correlated with reduced Shc phosphorylation and Ras activation (Macdonald et al., 2005a). While it is unclear whether the effects observed in replicon cells were caused by NS5A expression, it suggests that NS5A may disrupt the association of Grb2 and other adaptor proteins with the upstream receptor complex, thus blocking downstream Ras-Raf-MAPK activation at a very early step. The interaction between NS5A and Grb2, and the precise mechanism by which it blocks the downstream pathway need to be characterized in greater detail.

Grb2 and the downstream MAPK signaling pathways regulate many cellular processes such proliferation, gene expression, translational control, to name just a few. Thus targeting Grb2 and its downstream effectors through NS5A may have a significant influence on cellular functions and the HCV life cycle. In a follow-up study, it was found that NS5A inhibited the activity of AP1, a mitogenic and stress-activated transcription factor, through inhibition of the ERK pathway, and these effects were dependent upon the C-terminal Class II proline-rich motif that interacts with Grb2 (Macdonald et al., 2003). It was later shown in another study that an HCV replicon carrying a mutation within the C-terminal proline-rich motif lost the ability to block AP1 activation (Macdonald et al., 2005b). These results suggest that NS5A interaction with Grb2 may affect activation of the MAPK-dependent transcription factors and thus cellular gene expression. In addition, the ERK and p38 MAPK pathways also play a role in IFN signaling, by mediating serine phosphorylation of STAT1/3 transcription factors and contributing to maximal induction of IFN stimulated genes (He and Katze, 2002). Thus, in addition to its ability to modulate IFN responses directly by inhibiting the function of PKR, the ability of NS5A to modulate MAPK signaling may also contribute to the ability of HCV to modulate the IFN response. In addition, NS5A also blocked phosphorylation of eIF4E following EGF stimulation, which may provide a mechanism for down-regulation of eIF4E-dependent translation of capped cellular mRNAs, thus favoring cap-independent translation of HCV RNA (He et al., 2001).

In addition to Grb2, the C-terminal proline-rich motif of NS5A was also found to mediate interaction with the SH3 domain of several Src kinase family members, including Hck, Lck, Lyn, and Fyn (Macdonald et al., 2004). NS5A interacted with these Src family members in vivo and differentially regulated their kinase activity, inhibiting Hck, Lck, and Lyn while activating Fyn. Similar findings were noted in HCV replicon cells. However, the downstream effects as well as the physiological role of the NS5A interaction with these Src kinases remain unclear. It seems quite remarkable that one particular motif of NS5A is able to interact with so many cell signaling molecules. Despite all these interesting findings, the physiological role of the NS5A C-terminal proline-rich motif remains unknown, since mutation of this motif in an HCV replicon did not affect HCV RNA replication (Macdonald et al., 2005a). It may be possible that the conserved proline-rich motif is involved in other steps of the HCV infection life cycle, and this issue might be addressed with the recently development HCV infection system in cell culture. Alternatively, this motif and its interaction with cellular proteins might be required for successful HCV infection and viral pathogenesis in patients, but not for HCV life cycle in tissue culture, in which many aspects of the in vivo infection are missing.

NS5A has also been shown to interact with and modulate another pivotal cellular pathway, the PI3K-AKT cell survival pathway (Macdonald et al., 2005a). NS5A directly interacts with the p85 regulatory subunit of PI3K through the SH3 domain of p85. This interaction may involve either the N-terminal region, or a novel motif within the middle one-third of NS5A protein. NS5A was found to bind to heterodimeric PI3K in transient expression systems and enhanced the phosphotransferase activity of p110, the catalytic subunit of PI3K. The exact mechanism by which NS5A activates PI3K is not known, but NS5A expression increased the tyrosine phosphorylation of p85 PI3K following stimulation with EGF, indicating that NS5A interaction might facilitate the activation of PI3K by upstream signaling complexes. Along these lines, NS5A and p85 PI3K appeared to form a complex with Gab1, a cellular docking protein that provides a platform for the recruitment and activation of downstream signaling molecules in the vicinity of various growth factor and cytokine receptors (He et al., 2002). Stimulation of PI3K activity by NS5A results in increased phosphorylation and activation of AKT/PKB (He et al., 2002; Street et al., 2004). NS5A expression also modulated serine phosphorylation and function of the proapoptotic protein BAD, also a direct substrate of AKT. This indicates that NS5A might modulate host cell survival and contribute to HCV persistence by interacting with the PI3K-AKT cell survival pathway. Indeed, NS5A activation of the PI3K-AKT pathway correlated with the protection against apoptosis in NS5A-expressing cells or HCV replicon cells (Street et al., 2004). However, NS5A may also disrupt apoptosis through other mechanisms in these systems (see following parts in this section). In addition, expression of the HCV polyprotein in cells also activated the PI3K-AKT pathway, resulting in the modulation of two other AKT substrates, the Forkhead transcription factor and GSK-3β, indicating that HCV may affect multiple AKT-mediated pathways and biological functions (Street et al., 2005). Still, the downstream effects of the interaction between NS5A and the PI3K-AKT pathway are not completely understood and require clarification. Collectively, results from the studies reviewed here suggest a multi-faceted model of NS5A action in which NS5A is involved in the modulation of various cellular pathways. What is not so clear is which of these cellular pathways are physiologically important during HCV infection in vivo, and how the interactions between NS5A and multiple signaling pathways are coordinated and regulated during the HCV replication process.

Previous studies have shown that NS5A promotes cell proliferation resulting in cellular transformation through a PKR-dependent mechanism (Gale et al., 1999; Gimenez-Barcons et al., 2005). NS5A may also directly interact with the cell cycle control machinery. Several studies showed that NS5A repressed the expression of p21WAF1, a cell cycle regulatory gene (Ghosh et al., 2000b; Ghosh et al., 1999; Gong et al., 2004; Lan et al., 2002; Majumder et al., 2001; Qadri et al., 2002) resulting in increased cell proliferation and a transformed phenotype. The downregulation of p21 expression might involve direct NS5A interaction with SRCAP, a cellular transcription factor (Ghosh et al., 2000b), and in addition was suggested to be dependent on the tumor suppressor gene, p53 (Majumder et al., 2001). NS5A directly bound to and co-localized with p53 in the perinuclear membrane region, which may cause sequestration of p53 in this region (Lan et al., 2002; Majumder et al., 2001). NS5A inhibited the transcriptional activation activity of p53, resulting in inhibition of p21 expression, which is activated by p53 (Lan et al., 2002; Qadri et al., 2002). NS5A repression of p53 activity might involve additional factors in the p53 transcriptional activation complex. For example, NS5A was found to interact and co-localize with hTAF(II)32, a co-activator of p53. In addition, NS5A formed a heterotrimeric complex with TBP and p53 and inhibited the binding of these two proteins to their consensus DNA binding sequences (Lan et al., 2002; Qadri et al., 2002). These observations suggest that NS5A has the potential to interact with multiple cellular transcription factors and regulate the expression of cell-cycle control genes. However, in contrast to these experiments, two other studies showed that NS5A expression actually inhibited cell proliferation in various cell types, which exhibited a reduced S phase and an increase in the G2/M phase (Arima et al., 2001; Siavoshian et al., 2004). The underlying mechanism was suggested to be either p53-dependent induction of p21 (Arima et al., 2001), or through a p53-independent mechanism (Siavoshian et al., 2004). The underlying reason for the discrepancy in these results is not clear, but may be due to the different assay systems being utilized. Overall, the mechanistic details of how NS5A affects cell cycle control pathways are still not well understood and await further characterization in the HCV infection system.

Apoptosis is a proactive cell death process and in some cases is caused by viral infection. Prevention of host cell apoptosis may be beneficial to viruses by allowing longer periods of viral replication and persistence. It has been shown that NS5A could disrupt the apoptotic process through either PKR- or p53-dependent mechanisms (Gale et al., 1999; Lan et al., 2002). In addition, NS5A was able to inhibit apoptosis induced by treatment of human hepatoma cell lines with TNF-α. This effect correlated with a block in the activation of cellular caspases and downstream proapoptotic events (Ghosh et al., 2000a; Miyasaka et al., 2003). Interestingly, in transgenic mice expressing NS5A in the liver, TNF-induced apoptosis was prevented (Majumder et al., 2002). NS5A was found to physically associate with the TRADD signaling complex, which associates with the TNF receptor, and reduced the interaction between TRADD and FADD (Majumder et al., 2002; Park et al., 2002). So it seems that NS5A can block TNF-dependent apoptosis by associating with and disrupting the TRADD-FADD signaling complex.

In addition, it was found that NS5A expression inhibited TNF-induced activation of NK-κB, which is mediated by TRADD and TRAF2 (Majumder et al., 2002; Park et al., 2002). Consistently, NS5A directly interacts with and co-localized with TRAF2. The interaction was mapped to a.a. 148–301 of NS5A and required the TRAF-domain of TRAF2. However, NS5A did not block the recruitment of either TRAF2 or IKK-β to the TNF receptor complex, suggesting that NS5A may form a multi-subunit complex with at least TRAF2 and TRADD in the vicinity of TNF receptor (Park et al., 2003). Curiously, NS5A was also found to enhance TRAF2-mediated JNK activation by TNF-α (Park et al., 2003). It is unclear how the NS5A-TRAF2 interaction differentially modulates the NF-κB and JNK pathways. However, it is tempting to speculate that NS5A might disrupt host cell inflammatory and immune responses. What affect this might have in the context of HCV infection is not known. In addition, whether or not the NS5A-TRAF2 interaction is required for HCV RNA replication in cell culture requires further testing. Given that TRAF2 may also mediate cellular ER stress response and PKR-dependent NF-κB activation, it is possible that NS5A interaction with TRAF2 may also affect these cellular processes as well.

In another study, NS5A was able to antagonize sodium phenylbutyrate (NaPB)-induced apoptosis in hepatocellular carcinoma cells, a p53-independent process (Chung et al., 2003). NS5A was shown to co-localize and interact with Bax, a proapoptotic Bcl-2 family member, in the nucleus after NaPB treatment. Surprisingly, NS5A was found to contain a few Bcl-2 homology domains (BH3, BH1, and BH2; Fig. 1), which are domains found in Bcl-2 family members and mediate interaction between Bcl-2 proteins. BH3 and BH1 are in the N-terminal half of NS5A, while BH2 partially overlaps with the ISDR. A mutant of NS5A deleted for both BH2 and the putative NLS regions localized to the cytoplasm and disrupted its association with Bax. In addition, this mutant protein was no longer able to suppress NaPB-induced apoptosis. On the other hand, deletion of the NLS region alone resulted in a protein which still associated with Bax in the perinuclear region, but showed reduced association with Bax in the nucleus and reduced ability to block NaPB-induced apoptosis. These results suggest that NS5A may act as a Bcl-2 analogue and interact with Bcl-2 family members to block the apoptosis pathway, and this process may require the nuclear form of NS5A protein. As discussed previously, the biological function of the nuclear form of NS5A and the mechanism by which it is produced is not clear. Similarly, the role of the nuclear form of NS5A as it pertains to HCV infection requires additional experimentation.

Viral infection frequently results in activation of host cell defense mechanisms and stress responses, due to overexpression of viral proteins, stimulation of the innate immune responses pathways such as the IFN system, and disruption of normal cellular functions. In contrast to the above mentioned studies on the TNF receptor, Gong and colleagues found that NS5A expression activated the NF-κB and STAT3 transcription factors through oxidative or ER stress (Gong et al., 2001; Waris et al., 2002). NS5A seems to trigger oxidative stress by disturbing intracellular calcium pools, and the activation of NF-κB and STAT3 by NS5A is sensitive to inhibition by antioxidants and calcium chelators. Activation of the NF-κB pathway was also confirmed by microarray analysis of Huh7 cells expressing NS5A, since many NF-κB responsive genes were identified (Girard et al., 2004). The activation of the NF-κB pathway by NS5A may involve a novel mechanism involving tyrosine phosphorylation of IκB-α at two sites (Tyr42 and Tyr305) suggesting an alternative activation mechanism (Waris et al., 2003). Additionally, oxidative stress and activation of NF-κB have also been observed in HCV replicon cells, but it is unclear whether these effects are specifically caused by NS5A (Qadri et al., 2004; Waris et al., 2003). In addition, in NS5A-expressing transgenic mice, activation of the STAT3 transcription factor was also observed in the mouse liver (Sarcar et al., 2004). In this study, it was suggested that the activation of STAT3 by NS5A might involve the association of NS5A with the Jak1 kinase. It is unclear whether the NS5A-Jak1 association occurs during IFN signaling or whether this has an impact on IFN-induced antiviral responses. It is noteworthy that many of the cell culture-based studies reviewed in this chapter involve overexpression of NS5A and other HCV proteins at concentrations that are most likely higher than those in HCV-infected liver cells in patients. Thus, all these studies need to be considered in the proper context. Thus, future studies will more than likely be aimed at verifying these results in experimental systems that are more physiologically relevant as they become available.

In addition to the cellular signaling pathways discussed above, NS5A has also been reported to interact with a wide variety of cellular proteins. These NS5A-interacting proteins include karyopherin beta 3 (Chung et al., 2000), the adaptor protein amphiphysin II (Zech et al., 2003), the homeodomain protein PTX1 (Ghosh et al., 2003), and HSP27 (Choi et al., 2004), among many others [Table 1]. The exact physiological affects of these interactions require follow-up studies, but it seems likely that the range of cellular signaling pathways that are affected by NS5A is likely to expand.

Concluding Remarks

Tremendous progress has been made in our understanding of the biology of the NS5A protein. Recent biochemical and structural studies have given us great insight into the location of NS5A in various cellular compartments and the domain architecture of this protein. Various cellular binding partners have been identified and the affects of NS5A on various cellular signal transduction pathways continue to be an area of great interest. The role of phosphorylation of NS5A by host cell kinases continues to be defined. In addition, the advancement of the HCV replicon system has shed light on the physiological role of NS5A in viral replication. Despite this progress, several key questions remain. The precise role of the various forms of NS5A both in terms of subcellular localization and phosphorylation needs be systematically addressed. In addition, the role of other NS proteins in phosphorylation needs to be further refined and the clear identification of host cell kinases leading to both basal and hyperphosphorylation of NS5A requires additional work. One of the most exciting areas of research on NS5A is its interactions with host cell proteins and its ability to modulate host pathways. In most cases, the physiological role of these interactions needs to be studied in the context of viral replication. While we have learned much about the ability of NS5A to modulate the IFN response, more research is needed into its effects on other aspects of innate immunity. With the recent development of an HCV infection model, future work will most certainly be aimed at investigating the role of NS5A in other aspects of the HCV life cycle including viral entry and assembly. As new in vivo models evolve (see Chapter 12), the role of NS5A in virus replication in animal models will certainly be defined. Thus, future research should provide new clues as to the various functions of this truly remarkable multifunctional regulator.


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Copyright © 2006, Horizon Bioscience.
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