Sandbox Reserved 1088

Overall structure of DENV NS5 highlighting functional domains and major inter-domain interactions

NS5 is a 104 kDa, 900 amino acids long protein, making it the largest of the DENV proteins. NS5 is also the most conserved among the viral proteins with at least 67% sequence similarity among the four serotypes^[3]. According to structural and biochemical studies, two functional domains exist within NS5. These being a S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) domain which spans residues 6-262 and a RNA-dependent RNA polymerase (RdRp) domain spanning residues 273-883 that are connected to each other by a flexible linker domain 10 amino acids in length (residues 263-272)^[5].

Methyltransferase domain

The is involved in 5’ capping of the newly synthesized RNA - it catalyzes RNA cap methylation at both the N7 position on the guanosine and the 2’O on the subsequent nucleotide of the newly synthesized (+) RNA strand^[4][5]. The MTase domain contains five parallel beta sheets and within NS5, lies above the finger subdomain of RdRp in such an orientation that its catalytic core (K61-D146-K180-E216) and SAH binding pockets face away from the inter-domain interface ^[5].

RNA-dependent RNA polymerase domain

The is responsible for the replication and transcription of the viral genome and new viral RNA is synthesized without the need for a primer (de novo RNA synthesis). Like other polymerases, the RdRp is similar in that it contains finger, thumb, and palm subdomains as well as making use of a common catalytic mechanism for nucleotide incorporation involving two metal ions (zinc in NS5) located in the finger and thumb subdomains^[3]. Together, the thumb and finger subdomains form a through which the RNA template and nucleotides enter en route to the catalytic core within the palm subdomain. Inside the tunnel, a priming loop protrudes from a thumb subdomain towards the active site and is critical in de novo initiation^[3][4]. The RdRp is a feature unique to viruses and may be considered as a target for antiviral therapy.

Linker

The connects the two enzymatic domains and acts like a swivel allowing the domains to adopt various orientations relative to each other. Residues 263-266, located after the MTase C-terminus, are key in this regard. They fold into a short 3₁₀ helix (though other conformations are also thought possible) resulting in compaction or elongation of the polypeptide chain and allowing for various inter-domain interactions depending on viral requirements^[5]. Apart from this flexibility, it has been demonstrated that the linker residues (268-272) N-terminal to the RdRp region enhance stability and polymerase activity as evidenced by results showing superior activity in the complete NS5 protein as compared to the RdRp alone, further corroborating that MTase-RdRp inter-domain interactions are required to successfully maintain RNA synthesis, viral replication, and hence infectivity^[4][5][6].

Residues of the linker region

Inter-domain interface

Cluster 1

The majority of the stabilizing interactions within the inter-domain interface are polar in nature, resulting from contacts between charged side-chains as well as hydrogen-bonding mediated by trapped water molecules. Residues K95 and R352 form the major inter-domain polar contacts. The inter-domain is made up of two clusters with contributions from all three functional domains^[5].

Cluster 2

In the first cluster, interaction of linker residues with both RdRp and MTase domains is observed. Specifically, linker residue E267's side chain hydrogen bonds with residues Y119 and R262 of the MTase domain while E269 forms a salt-bridge with R361 of the RdRp domain. Polar interactions between residues from the MTase domain (K95 & K96) and the RdRp domain (E296-K300) also play an important role in the cluster.

The second cluster is based at residues (F348-K357) in the RdRp domain where guanidinium of R352 interacts electrostatically with a number of residues in the MTase domain (E67, E252, Q63). A salt-bridge is also formed between K357 and D256. Though hydrophobic interactions are not as numerous as hydrophilic interactions, they do exist as stacking interactions, namely between W64, R68, F348, and P582^[5].

Through mutation studies in DENV-4, residues K95 and R353 have been specifically implicated in critical but non-enzymatic roles in virus RNA replication and infectivity while Y119 has been shown to be necessary for MTase activity and therefore a requirement for viral replication. Because of a majority of polar interactions as well as a small inter-domain buried surface area (1502 Å), it has been suggested that the two enzymatic domains may be able to associate and dissociate from each other with a relatively small energy penalty. Additionally, this dynamic interaction may also lend itself to facilitating the recruitment of other viral and host proteins as part of the replication complex^[5].