Since NS4B is a multipass transmembrane protein, its putative TMDs contribute to at least one-third of the protein size. This implies that in addition to anchoring NS4B to host membranes, the TMDs could actively participate in the folding of the protein via intramolecular interactions. Further, as a scaffolding protein, NS4B may need intermolecular interactions in the context of host membranes (i) to recruit host and viral proteins to the RC and (ii) to oligomerize so as to form the typical NS4B foci. Thus, we have hypothesized that exchanging the NS4B TMD sequences between the HCV Con1 and JFH1 strains could lead to a defect in JFH1 virus production and virus genome replication. In light of the findings that the TMD sequences are not identical for different HCV genotypes, the small differences in amino acids could give rise to incompatibility between the NS4B proteins. Further, the importance of the TMDs in NS4B function is illustrated by the findings that several drug resistance mutations leading to NS4B-induced apoptosis, or HCV replication, can be mapped to the NS4B TMD1 and TMD2 sequences (58
To test the prediction described above, we engineered JFH1 chimeric RNAs containing all or parts of the Con1 NS4B protein. In this report, we have identified the TMDs as major determinants of NS4B function in HCV replication. The TMDs of Con1 and JFH1 are generally incompatible, implying that the NS4B TMDs from different genotypes may not bind efficiently to each other or do not allow the chimeric NS4B protein to interact effectively with other replicase proteins. Our finding that a mutation in the Con1 TMD2 sequence restored the replication efficiency of the defective TMD2 chimera further supports this interpretation. More importantly, we have identified two putative helix dimerization motifs in the TMDs: the S/T-like motif in TMD1 and the GXXXG-like motif in both TMD2 and TMD3. Mutagenesis of residues in the putative dimerization motifs in TMD1 and TMD2 generally resulted in a loss of JFH1 replication, suggesting that these motifs might play a role in NS4B interactions in the context of the host membranes. These interactions may contribute to NS4B stability, as evidenced by the fact that two mutations in the TMD2 sequence led to the destabilization of the mutant proteins. Finally, we have shown that the JFH1 chimera with the Con1 NS4B CTD is defective in virus production, not replication. This finding confirms the previous report by Jones et al. (33
) indicating that NS4B plays a role in virion production.
In contrast to the roles of the NTD (9
) and CTD (3
), we know very little about the role of the TMDs in NS4B function. Few studies concerning the NS4B TMD region have been reported previously. For example, Lindström et al. (42
) have used alanine scanning mutagenesis to demonstrate that certain residues in the loop regions of TMD1-2, TMD2-3, and TMD3-4 are required for HCV replication, but the underlying mechanism is still unknown. Einav et al. (17
) reported that NS4B GTPase activity is required for HCV genome replication but is not associated with NS4B foci, the platform for HCV RC assembly (3
). The exact role of the TMD helices has been overlooked in part because of the assumption that these domains merely insert NS4B into host membranes. However, TMDs are now recognized as major instigators of protein-protein interactions, thanks to biochemical, biophysical, and genetic analyses of these domains (8
). Some specific motifs have been found responsible for TMD helix homo- or heterodimerization. GXXXG, the most widespread motif, was initially found in the human erythrocyte sialoglycoprotein glycophorin A (GpA) TMD (47
). It was later identified in several host and viral integral membrane proteins whose TMDs have been reported to homo- or heterodimerize (8
). The GXXXG motif tends to induce membrane helix interactions because its short side chain (—H) promotes helix-helix association via van der Waals forces and H bond interactions (39
). Interestingly, two recent studies have shown that the GXXXG motif in the HCV E1 TMD sequence is required for E1–E2 heterodimerization (13
), an event that may be linked to HCV entry and budding from the cells.
In NS4B protein, a GXXXG-like motif was found in TMD2 (G125
) and TMD3 (G143
). Since exchanging TMD2 between Con1 and JFH1 resulted in a significant decrease in JFH1 replication, we chose to investigate the role of the putative GXXXG motif in NS4B function in light of the findings that the nonconservative changes in amino acid sequences in TMD2 play little to no role in the incompatibility between the Con1 and JFH1 sequences. Whereas the G125A mutation led to a severe defect in replication efficiency, the G129A mutation did not. If Gly125
are part of a GXXXG-like motif, then the WT phenotype of the G129A mutation could be explained in part by the facts that (i) both Ala and Gly residues have smaller side chains than Leu, allowing close packing of the helices to occur and (ii) two HCV genotypes have Ser129 or Ala129 in NS4B TMD2 (), suggesting that this position can tolerate changes with residues that have small side chains. In contrast, the replication-defective G125A phenotype may indicate that this position cannot tolerate small residues such as Ala in TMD2 (47
). Indeed, variants of the GXXXG motif, including GXXXA, AXXXG, SXXXG, and AXXXA, have also been reported to mediate membrane-spanning domain interactions (6
). While mutation of the Gly to residues with a bulkier side chain, such as Leu, will definitely destroy the GXXXG-mediated TMD interactions, the impact of a G-to-A or G-to-S mutation may depend on the context of the motif.
The GXXXG motif predicts that replacement with bulky residues, such as Leu (with large side chains), will reduce van der Waals and H bond interactions between interacting TMDs. Thus, the findings that the G125L and G129L mutations lead to severe decreases in JFH1 replication efficiency suggest that Gly125
may be part of a GXXXG-like motif. However, G129
is also the first residue of a previously reported Walker A motif (G129
). Therefore, the phenotype of G129L mutation may just reflect the loss of NS4B GTPase activity. The finding that G129A mutant RNA replicates as well as JFH1 RNA may suggest that Gly129
has a dual role in NS4B function: in addition to its reported role in nucleotide binding, we propose that Gly129
is part of a GXXXG-like motif. Future experiments will seek to (i) identify NS4B TMD2-interacting partners, (ii) test whether the conserved Gly residues in both TMD2 and TMD3 are functionally important, mimicking the GXXXG motif, and thus may play a role in protein-protein interaction, and (iii) determine how mutations in this motif affect NS4B properties, including GTPase activity and RNA-binding ability.
Our study has also identified an S/T cluster-like motif in the NS4B TMD1 sequence. This motif is less common among integral membrane proteins, but it has been found in TMD libraries of model membranes (15
). Indeed, owing to their uncharged but polar properties, Ser and Thr residues in the hydrophobic environment of membranes tend to be engaged in helix-helix interactions via H bonds (15
). Replacing these Ser and Thr residues with Ala should have no negative impact on NS4B function unless these changes disrupt H-bonding interactions in the side chains (15
). Indeed, except for TMD1 residue Ser91
, mutagenesis of residues in the putative S/T cluster [S83A, TS(87/88)AA, TT(94/95)AA] led to a significant decrease in HCV replication. These data imply that some residues in the S/T cluster-like motif might be engaged in interaction, probably via their —OH group in the side chain. Thus, future studies will seek to (i) reveal the putative NS4B TMD1-interacting partner(s), (ii) define the role of the S/T cluster-like motif in this activity, and (iii) investigate how mutations in this putative motif affect NS4B activities.
As mentioned above, the TMD sequences may participate in (i) the NS4B interactions with host and viral proteins, (ii) the folding of NS4B protein, and/or (iii) NS4B oligomerization (74
). The replication defect in the JFH1 chimeras containing the entire Con1 NS4B or its TMD region may be due in large part to the disruption of the interaction between NS4B and host/viral proteins. As with the smaller TMD helix swaps, since both TMD2 and TMD3 have GXXXG-like motifs in their sequences, we postulated that Con1 TMD2 and TMD3 would be more compatible via interaction of their helices, stimulating the replication of the JFH1 chimera. We predicted that, if this is correct, a JFH1 chimera containing the Con1 TMD2-3 sequence would replicate better than the chimera with the Con1 TMD2 sequence alone. Surprisingly, Luc-J/C1-B(2-3) replication was severely impaired relative to that of Luc-J/C1-B(2) (). Further, Luc-J/C1-B(2-4) replicated better than Luc-J/C1-B(2-3) (). These findings suggest that the NS4B TMDs are perhaps engaged in complex intra- and intermolecular interactions, which remain to be uncovered. For visualization of the TMDs, we performed homology modeling to predict the conformation of the JFH1 NS4B TMD region that has an amino acid sequence homologous to that of known protein structures (see the supplemental figures at http://bmb.psu.edu/directory/kvk10
This model shows Gly129 at the interface between the endoplasmic reticulum membrane and the cytosol. However, this is a static model, which does not take into account any potential movement that could occur in NS4B TMDs. Considering that (i) the NS4B molecule has two proposed amphipathic helices and that (ii) membranes are dynamic, the proposed model may represent an “off” state of the TMDs. Thus, if our model is true, conformational changes must occur for NS4B enzymatic activity (GTPase) to be achieved. Although this model needs to be confirmed using structural approaches, it nevertheless predicts putative intramolecular NS4B interactions involving the TMDs. Further, the findings that the defect in J/C1-B and J/C1-B(2) chimera replication can be rescued by a second-site mutation in the NS4B TMD2 sequence suggest that at least the TMD2 region is engaged in genetic interactions. Whether such interactions are intra- or intermolecular will be the subject of future studies.
NS4B expression results in the formation of foci that serve as a platform for the recruitment of the HCV RC (24
). To understand the mechanism underlying the defect in the chimeric or mutant JFH1 replicon RNAs, we examined the subcellular distribution of various JFH1 NS4B proteins. As expected, all the chimeric NS4B proteins displayed WT-like foci (data are shown only for J/C1-NS4B and J/C1-B), suggesting that these chimeric proteins might be defective in their ability to (i) recruit host/virus factors to the HCV RC, (ii) bind to nucleoside triphosphates (GTP/ATP), or (iii) bind to the HCV RNA.
Further, whereas most of the mutant proteins displayed a WT-like NS4B subcellular distribution, the NS4B F118A and G125L mutations resulted in significant disruption of NS4B foci (). Additionally, several attempts to investigate the membrane association of NS4B F118A and G125L proteins were unsuccessful, whereas the binding of mutant G125A, J/C1-B(1), and J/C1-B(2) NS4B proteins () to host membranes was not significantly altered from that of WT NS4B. These findings suggested that either the mutant NS4B F118A and G125L proteins are unstable or the constructs resulted in low transfection efficiency of these plasmids. However, the findings from pulse-chase and immunoblot experiments () are consistent with the interpretation that the F118A and G125L mutations destabilize NS4B protein. The F118A phenotype may be explained by a disruption of the TMD2 interaction(s) required for NS4B folding. The phenotype of the G125L mutant was surprising, since the G125A mutation had no significant effect on NS4B stability. Since the G125A and G125L mutations negatively impact replication, we interpret these results to mean that the G125A mutation disrupts a single intra- or intermolecular TMD2 interaction, whereas the G125L mutation may have a more global effect on such interactions. However, we do not rule out the possibility that the F118A and G125L mutations can destabilize the TMD2 helix, thus rendering NS4B unstable. Studies are under way to test whether such mutations destabilize NS4B in the context of the replicase complex.
In conclusion, this study has revealed, for the first time, the importance of the TMDs in the NS4B role in HCV replication. Future efforts will focus on using genetic, biochemical, and biophysical approaches to further investigate the contribution of the TMDs to NS4B function in the HCV life cycle.