Conserved GXXXG- and S/T-Like Motifs in the Transmembrane Domains of NS4B Protein Are Required for Hepatitis C Virus Replication

Abstract

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra-and intermolecular interactions required for NS4B structure/ function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virus responsible for 170 million cases of chronic infections worldwide. HCV is the only member of the genus Hepacivirus in the family Flaviviridae (51, 63), which includes other human pathogens, such as West Nile virus and dengue virus. Translation of the virus genome yields at least three structural proteins (core, E1, and E2), the highly hydrophobic p7 peptide, and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The NS proteins, including NS3 to NS5B, are sufficient to promote HCV replication in vitro (10,43). However, with the advent of the HCV cell culture system, many of the NS proteins (NS2, NS3, and NS5A) have been reported to play an active role in HCV assembly (5,46,50,69,73), further supporting the idea that the NS proteins in general have multiple functions in the HCV life cycle.NS3 is illustrative of multifunctionality. Its N-terminal serine protease activity is responsible for processing the NS proteins into their mature forms, whereas the C-terminal helicase activity may be required for the unwinding of HCV RNA (35,66). Similarly, NS4A is a cofactor of NS3 serine protease; it also assists NS3 in binding to host membranes (72) and facilitates the association of NS3 with the HCV replication complex (RC). NS5A may have multiple functions, including inhibition of the interferon response to virus infection and HCV RNA ...

Targeting Cellular Squalene Synthase, an Enzyme Essential for Cholesterol Biosynthesis, Is a Potential Antiviral Strategy against Hepatitis C Virus

“…We found small intense foci that were detected with an anti-HA antibody in non-drugtreated cells (Fig. 10C) and are similar to the NS4B foci previously reported (58,60,61). The foci were not detected in Huh-7.5.1-8 cells transfected with a backbone plasmid (Fig.…”

Modulation of Hepatitis C Virus Genome Encapsidation by Nonstructural Protein 4B

“…The plasmid pLuc-JFH1, which contains the T7 promoter sequence fused to nucleotides (nt) 1 to 389 of the JFH-1 consensus sequence, followed by the firefly Luc gene, the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), and the nucleotides spanning from the beginning of the NS3 gene to the 3= nontranslating region (NTR) of JFH1, was constructed according to methods previously described (46,47). Overlap extension PCR was used to construct the full-length genomes containing the chimeric C-terminal domain of the NS4B gene, where the JFH-1 sequences were replaced with either Con1 (J/Con-C) or H77 (J/H-C) sequences, and was performed according to the methods previously described by Han et al (33). The various subgenomic Luc replicons were constructed by directly replacing the fragment between the EcoRI site and the SpeI site of their corresponding full-length constructs with EcoRI-and SpeI-cut fragments from pLuc-JFH1.…”

“…Blunt-end ligation was used to introduce the single point mutations into the JFH-1, J/Con-C, or J/H-C genome, as previously described (33). Since the single mutations were engineered by the same approach, we use the pJ/Con-C N216S (NS4B) vector to illustrate how the constructs were made.…”

“…The transmembrane region of NS4B is composed of at least four membrane-spanning domains (transmembrane domains [TMDs]) and was recently shown to contain protein-protein interaction motifs crucial for HCV genome replication (33). The N-terminal domain (NTD) of NS4B was reported to have dual-membrane topology (32,34), whereas the C-terminal domain (CTD) is located on the cytosolic side of the ER membrane.…”

“…We have recently demonstrated that swapping NS4B CTD sequences between HCV JFH1 (infection results in a relatively high virus titer) (41)(42)(43) and HCV Con1 (infection gives rise to a low virus titer) (44) leads to an attenuation of the JFH1 virus titer (33) with a negligible impact on genome replication. This finding suggests that NS4B is a multifunctional protein with distinct roles in both replication and virus production.…”