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Dengue Serotype 3 RDRP NS5 analysed at 1.85 A resolution

Prevalance

Dengue fever is a mosquito-borne disease caused by four viruses (DEN-1, DEN-2, DEN-3, DEN-4). Dengue viruses, interestingly, are quite comparable to the viruses which cause West Nile infection. The symptoms of this virus tend to appear 3-14 days after an individual is bitten by an infected mosquito. While Dengue fever is not typically fatal, the symptoms can be quite intense. Further, these symptoms may include sudden fever, intense headache, eye pain, bleeding, and muscle and joint pain. Additionally, vomiting and diarrhea may arise in a host after infection. These symptoms tend to last between 4-7 days of onset. A more severe form of Dengue fever is associated with hemorrhagic fever. This type of fever leads to damage to blood cells in an infected host like the hemorrhagic fever associated with Ebola. However, the emergence of hemorrhagic fever is typically due to a host being reinfected with a different serotype than they were previously infected with. For example, hemorrhagic fever may occur in an individual who is infected with DEN-2 a few months after being infected and treated for DEN-1. Dengue fever, while typically brought back to the USA from travel, does have an impact on the US population. Moreover, this virus caused 332 dengue cases in the US in 2020 alone. Globally, however, the 332 infections seen in the USA pale in comparison to the global impact of this virus. Moreover, it is anticipated that 390 million infections occur around the world each year. Of these 390 million infections, around 500,000 cases lead to a severe infection or subsequent dengue hemorrhagic fever. This virus is estimated to lead to as high as 25,000 deaths annually. There is, thankfully, a vaccine for Dengue. The vaccine, Dengvaxia (CYD-TDV), developed by Sanofi has an efficacy of 76%. This data infers that the vaccine certainly provides a layer of protection between a human host and the virus. However, in general, this vaccine is not recommended to individuals who have never been infected by Dengue. This is because if an individual who has recently been vaccinated ends up getting infected shortly after the vaccination, they are at high risk for hemorrhagic fever associated with Dengue. The RNA classification of the virus is single-stranded positive-sense ribonucleic acid composed of 10,700 bases. This virus belongs to the Flavivirus genus which includes other viruses such as yellow fever, Zika, and West Nile.

Function

The function of RNA-dependent RNA polymerases (RDRP) is viral genome replication and transcription. Furthermore, RDRPs can catalyze the process of making rRNA as well which also impacts downstream translation of functional proteins. This in mind makes a promising drug target since if RDRPs are inhibited replication and all subsequent processes leading to protein synthesis are effectively shut down. Henceforth, the genome of an infectious virus can have replication of the virus shut down as the viral RDRPs associated with the virus and these RDRPs do not have human homologs. Thus, shutting down the replication of the virus does not mean shutting down human replication.

Conservation of RDRP Hand Structure

Currently, there are two proposed drug candidates, verified through crystal structure, for Dengue serotype DEN-3; the two proposed small molecules for inhibition of Dengue RDRP are NITD-640 and . The finger subdomain in Dengue RDRP is made up of . The "fingertip region" has been shown to be more mobile than the palm and thumb subdomains. Consistent with other viral RDRPs there is an extended fingertip region in Dengue RDRP. Helix-6,14 and 15 are shaped by solvent-exposed residues which lead to the formation of a concave surface at the finger base. Unlike other RDRPs, however, there is an N-terminal segment of 35 amino acids added which is composed of helix-1 and strand-1 which connect to helix-two of NLS which is buried in the thumb domain. Two loop regions

link the finger and thumb domains. This linkage likely leads to confirmation changes between the two domains, thus hindering independent movement between these regions. In other RDRPs the fingertips of loops twist away from the active site, but in Dengue RDRP loop 3 regulates access of ssRNA substrate at the entrance of the template tunnel. The overall sequence conservation of the finger region of Dengue finger regions is quite low compared to other viral RDRPs.

The palm domain is made up of with the addition of a small antiparallel beta-strand platform and eight alpha-helices. The palm domain is understood to be the most structurally conserved among all known RDRPs. This demonstrates the low evolutionary pressure to change the catalytic site of this protein. Moreover, the active site residues have been demonstrated to superimpose very tightly across other RDRP models. The catalytic site made up of strand-4 and 5 composed of Asp-663 and 664 has strands that are two times shorter than those found in other RDRPs. The connection between motif B and C is also more elaborate in flavivirus than in another genus. Thus, there are key signatures in the flavivirus active site of RDRP which make it unique to this genus. Crystals of this protein were soaked with Magnesium Chloride which demonstrates a magnesium ion is present near its expected catalytic position (Nascimento et al., 2021). This ion is coordinated by

The thumb domain of Dengue's RDRP consists of This domain forms at the C-terminal end and is the most structurally variable among all known RDRP structures. However, it does conserve two sequence conserved motifs. Motif E wedges a beta-sheet between itself and many thumb domain alpha-helices in the thumb domain. The loop link between helix 21 and 22 takes on a unique conformation in contrast to other viral RDRPs in that it forms a beta-hairpin. This loop link in conjunction with the fingertip region helps form the shape of the RNA template tunnel. The priming loop is formed by residues 782-809. Internal intra-loop interactions stabilize this region by forming hydrogen bonds and bonds between the residues Thr-794 and Ser-796 with a salt bridge amongst Glu-807 and Arg-815 along with stacking between Arg-749 and the indole ring of Trp-787. There are two zinc ions in this RDRP. The ions likely contribute to structural stability near motif E. Additionally, this binding pocket is near the functionally important residues Ser-710 and Arg-729. Due to this, it is postulated that these ions could cause the regulation of conformational switches within the thumb domain. It is worth noting that there is high sequence conservation of the metal-binding residues between the flavivirus RDRPs (Nascimento et al., 2021). While no crystal structures demonstrate direct RNA binding with Dengue RDRP, it is observed that this structure has dimensions that would allow access to only ssRNA at the active site.

Orientation of RNA Template

RNA synthesis begins with NS5 attaching to the 3’ end of the template strand (Nascimento et al, 2021). Simultaneously, GTP and ATP are introduced to the active binding site to facilitate Watson-Crick base pair formation with the conserved U and C bases at the 3’ end (Nascimento et al, 2021). Orientation of the ssRNA template shows the sugar-phosphate backbone of the strand “pointing away from the binding groove of the fingers subdomain” (Yap et al, 2020). The ssRNA forms an electrostatic interaction with the positive charge of the finger subdomain (Yap et al., 2020). This interaction stabilizes the 3’ end at the active site cavity (Yap et al, 2020). The palm subdomain active site residues involved in RNA template stranding are

Asp533 and Asp664. It is at the initiation step that Mg2+ coordinates the initial dinucleotide primer (pppAG) synthesis formed by a guanine-α-phosphate and an alcohol group from the adenine ribose (Nascimento et al, 2021). In the pre-initiation step, the exit tunnel for the RNA was closed, but this opens up at the onset of initiation to “ensure the process of RNA polymerization”. The Zn2 ion likely plays a role in the stability of DEN polymerase. This pocket is located near Ser-710 and Arg-729. Furthermore, in the priming loop 3'dGTP is coordinated by Ser-710, Arg-729, and Arg-737. These residues are indeed structurally conserved residues across all positive-strand RNA viruses. These residues are known to initiate replication through a . The zinc ion in metal binding pocket one is believed to catalyze this priming reaction due to its proximity to key residues. Additionally, loops L1, L2, L3, the linker alpha21-alpha22 and the priming loop enclose the catalytic active site and form the shape of the template tunnel. The tunnel is around 30 angstroms deep and 24 angstroms wide. These dimensions suggest that only around five to seven nucleotides fit into the active site. Moreover, several loops likely take a different conformation to make room for the translocation of a duplex created through the synthesis of a new RNA strand and template. It is believed that motion of helices in the finger domain accompanied with the closing of the active site around the primer-template occurs to secure the template strand for replication.