Hepatitis C virus (HCV) which belongs to the family flaviviridae, causes chronic liver disease, liver cirrhosis and hepatocellular carcinoma in humans. Non-structural protein 5A (NS5A) of HCV is involved in regulating HCV RNA replication and evading the interferon induced antiviral response by interacting with human protein kinases as the viral genome does not encode any kinase.
Structural information of NS5A at atomic level is known only for the N terminal part of the protein. Residues 5–25 of NS5A adopts α-helical conformation which is amphipathic and is embedded in a phospholipid bilayer on one side and the other side has conserved polar residues. Residues 36–198 of NS5A adopt a novel zinc coordination motif which forms a homodimer and is organized into different forms to perform different roles such as viral replication and viral assembly[6, 7]. Both the regions of NS5A have been shown to be critical for viral replication[5, 6]. Experimental work have shown that for the rest of the protein whose structure is currently unknown it is natively unfolded, lacks secondary structural elements, is less hydrophobic, has high content of positively charged residues, has low complexity regions and many phosphorylation sites which predominantly occurs in intrinsically disordered regions of the protein[9–11]. Due to these attributes, NS5A which is natively unfolded, attains a stable structural form when it is bound with a protein. The natively unfolded nature of NS5A makes it capable of interacting with many human as well other non-structural proteins of HCV thereby carrying out multiple functions.
NS5A interacts with different human kinases namely, casein kinase 1α (ck1α) and Protein kinase R (PKR). In the present study, the critical kinase and NS5A residues involved in interaction have been predicted by protein structure modeling and sequence analysis.
The Casein kinase 1α – NS5A interaction forms a transient enzyme-substrate complex. Several amino acids in NS5A get phosphorylated by different human kinases. Varying levels of NS5A phosphorylation modulates its interaction with host and other viral proteins during viral replication. Phosphorylation of NS5A serves as a regulatory switch between viral replication and RNA translation and / or packaging. NS5A phosphorylation is observed among the flaviviridae family which points out its functional significance for the viral life cycle.
Apart from the viral protein NS5A serving as a substrate to CK1α, one of the other substrate for CK1α is tumour suppressor p53 transactivation domain. DNA virus induced stress to human cells leads to phosphorylation of serine 20 of p53 transactivation domain by CK1α. This phosphorylation enables the binding with a co-activator (p300) and stimulates tumour suppressor p53 function.
Casein kinase 1 is a serine / threonine protein kinase which is ubiquitously expressed in all the tissues and cellular compartments of eukaryotic organisms. Human casein kinase 1 phosphorylates substrates which are involved in the control of cell differentiation, proliferation, chromosome segregation and circadian rhythm. The catalytic domain of casein kinase 1 is about 300 amino acids long. The catalytic domain of casein kinase 1 is made up of 2 lobes with a cleft between them for the substrate peptide to bind.
Human ck1 shows high sequence identity to the other members of casein kinase 1 family. The crystal structure of casein kinase 1, from Schizosaccharomyces pombe, bound to ATP closely resembles both cAPK (cAMP dependent kinase) and Cdk2 (cyclin dependent kinase), whose crystal structures have been solved along with substrate peptide and ATP.
Even though NS5A is phosphorylated at several residues by many human kinases, clear one-to-one mapping between phosphosite in NS5A and its corresponding human kinase is not completely known. However the human CK1α phosphorylating sites have been mapped to Ser 232 of NS5A and, therefore, phosphorylation by CK1α is the focus of the current study. In the present work, structural model of human CK1α bound to NS5A peptide of HCV containing the phophoacceptor serine 232 has been built using available crystal structures of other kinase-substrate/pseudosubstrate complexes. The aim of building the model of the complex is to understand the structural basis of the NS5A peptide recognition and identification of NS5A interacting ck1α residues. The analogy of ck1α – substrate recognition and interaction is also shown by considering the example of another substrate of ck1α - tumour suppressor p53 transactivation domain, whose crystal structure is known.
NS5A also interacts with another protein kinase PKR during the life cycle. NS5A does not get phosphorylated by PKR but only forms a protein-protein complex.
Interferons lead to expression of IFN effector proteins like the double-stranded RNA-activated protein kinase (PKR). During viral infection, PKR becomes activated upon viral dsRNA binding, dimerisation and trans-autophosphorylation. PKR in its activated state phosphorylates the eukaryotic translation initiation factor 2 (eIF-2α), thereby inhibiting cellular protein synthesis and blocking viral replication.
In the recent past, triple therapy, which includes pegylated interferon along with ribavirin and a HCV protease inhibitor, has shown promising results with high rates of sustained virological response compared to other treatments currently available[23–25]. Combined pegylated interferon, ribavirin therapy provides ~45-50% sustained virological response in genotype-1 infected patients[26–28] while when interferon alone is administered only ~30% of genotype 1 infected patients seem to respond to the therapy. This is mainly because a region in NS5A [interferon sensitivity determining region-ISDR (237–302)] interacts with PKR [dimerisation domain (244–296)], thereby preventing PKR dimer formation and activation. Additionally, another region in NS5A, referred as the Variable-region 3 (V3) (381–407) is located downstream to ISDR which is also involved in the interaction and inhibition of PKR. Interestingly, more than 60% of genotype 2 and 3 patients respond well to the interferon therapy. One of the reasons for this is that NS5A 2a/3a are unable to bind and inhibit PKR efficiently.
An attempt has been made to narrow down the region in NS5A and predict critical residues in NS5A 1b responsible for interaction with PKR by two independent approaches. Firstly, due to putative shared binding region of NS5A 1b and PKR protomer with another PKR protomer, one can hypothesize that structural features of PKR dimer interface and PKR interacting region in NS5A 1b would be similar. The unstructured region of NS5A 1b[9–11] could attain a stable structural form and have similar binding surface like that of PKR protomer as it binds to the other PKR protomer. Some of the critical PKR interacting residues of NS5A has been identified using this approach.
Further, as the binding of NS5A with PKR is primarily genotype specific[14, 29, 30], evolutionary trace analysis[31, 32] has been used to identify amino acid residues in NS5A specific for genotype 1 that interacts with PKR and residues in NS5A specific to genotype 2A and 3A which is potentially incapable of binding and inhibiting PKR efficiently.
Details of the NS5A and human kinase interacting residues shown in this study can provide insights to design antivirals to inhibit these interactions.