Computational Analysis of the Hepatitis C Virus Non-Structural Protein 4B and Functional Implications

Introduction: NS4B is a multifunctional protein, involved in membranous web formation and regulation of signal transduction cascades. The protein is located with the endoplasmic reticulum membrane. Four to five transmembrane domains (TMDs) were discussed for the membrane topology. N- and C-termini are most probably oriented towards the cytoplasm. Although the flaviviridae family has a similar genetic organisation, a comparison of HCV NS4B to other members of the family is still missing. Methods: We used bioinformatic approaches to analyse structure and function relationships in HCV NS4B. Using the BLAST suite of programs (NCBI-NR and UniProt), we extracted a set of candidate sequences. Representative sequences were selected for each clade of the phylogeny. Subsequently, multiple alignments were generated and hand-curated with CLUSTAL W. To get reliable overall results, we used secondary structure and transmembrane prediction methods, as well as consensus procedures. Results: We could confirm a central non-globular membrane portion with four to five TMDs from a N- and C-terminal part with non-transmembrane helices. The central part showed new functionally-relevant sequence motif. The N-terminal part of NS4B was the most heterogeneous in phylogenetic and sequence analysis of flavi-, pesti-, GB- and hepaciviruses, with GB-viruses more closely related to hepaci- than to other flaviviruses. A heptad repeat within a leucine-rich amphipathic helix has been characterized, suspected as functionally important interaction site. The C-terminal part shows significant similarities in sequence and secondary structure throughout flaviviridae. Conclusions: NS4B N- and C-terminal parts seem to be functionally important, whereas the central protein portion serves as membrane anchor. A N-terminal leucine zipper motif is unique in hepaciviruses and probably important to interact with the basic leucine zipper (bZIP) domain of CREB-RP/ATF6β, a human activating transcription factor, stimulated upon ER-stress. That way HCV could be able to interfere and modulate ER-stress induced gene expression patterns. Site-directed mutational studies are needed to identify crucial positions within the NS4B bZIP motif and study effects on viral replication and cellular transformation.

Keywords: NS4B, flaviviridae, insilico analysis, phylogenetics