ReviewA structural view of the RNA-dependent RNA polymerases from the Flavivirus genus
Introduction
The flaviviruses form the largest genus among the Flaviviridae family. According to the International Committee on Taxonomy of Viruses (ICTV, www.ictvonline.org), currently there are 53 virus species being assigned in the Flavivirus genus. The well-known mosquito-borne flaviviruses include Yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Dengue virus (DENV), affecting at least one-third of the world population, mostly in tropical and subtropical regions. Since 2015, the Zika virus (ZIKV), being a phylogenetic close relative to DENV, has raised a global concern due to its capability in maternal-fetal transmission (Lazear and Diamond, 2016). Although being less transmissible, the tick-borne flaviviruses such as Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) can also cause severe neurological or hemorrhagic diseases. Flaviviruses without known vector such as Yokose virus (YOKV) and Modoc virus (MODV) also exist.
Despite the diverse mechanisms of transmission, the flaviviruses share a common genome organization. The single-stranded RNA genome is positive sense and is about 10.2–11 kilobases in length (Chambers et al., 1990, Westaway et al., 1985). It bears a type 1 cap at its 5′ end and does not have a 3′ poly-adenine tail (Bisaillon and Lemay, 1997, Cleaves and Dubin, 1979) (Fig. 1A). The genome contains a single open reading frame (ORF) that encodes a polyprotein of about 3400 residues and 5′ and 3′ untranslated regions (UTRs) bearing structured elements regulating viral genome replication (Filomatori et al., 2006, Zhang et al., 2008). The polyprotein is processed by viral and host proteases to yield ten proteins (Apte-Sengupta et al., 2014, Brecher et al., 2013, Zhang et al., 2016). Among these, three structural proteins C, prM, and E are responsible for viral capsid and envelope construction (Mukhopadhyay et al., 2005), and the seven non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 assemble into a complicated genome replication complex that resides in membranous substructures in the endoplasmic reticulum (Romero-Brey and Bartenschlager, 2016, Welsch et al., 2009). Being the largest and most conserved flavivirus protein, NS5 has a very unique organization with a capping related methyltransferase (MTase) and the central replication enzyme RNA-dependent RNA polymerase (RdRP) naturally fused through a 10-residue flexible linker (Khromykh et al., 1999, Koonin, 1993, Lu and Gong, 2013) (Fig. 1B). In this review, we present our current understanding of Flavivirus RdRPs primarily from the structural perspective. The overall RdRP architecture, key motifs and functional elements related to polymerase catalysis, and the autoregulation of RdRP from its natural partner MTase are relatively discussed in detail, while the RdRP regulations by other viral and host factors, or other processes may be mentioned but are not comprehensively presented.
Section snippets
The architecture of flavivirus RdRP and the priming element for de novo initiation
The earliest crystal structures of flavivirus RdRP (of WNV and DENV) were solved in 2007 (Malet et al., 2007, Yap et al., 2007), and structurally they are quite homologous to their Flaviviridae counterparts such as the NS5B proteins encoded by the Hepatitis C virus (HCV) and the Bovine viral diarrhea virus (BVDV) (Ago et al., 1999, Bressanelli et al., 1999, Choi et al., 2004, Lesburg et al., 1999). Similar to RdRPs encoded by all other RNA viruses, the RdRP module of NS5 contains a core region
Seven catalytic motifs common to all viral RdRPs
All viral RdRPs share seven catalytic motifs A through G (Bruenn, 2003, Gorbalenya et al., 2002, Poch et al., 1989). Both sequence and structure conservations contribute to the identification of these motifs as only motifs A, B, C, and F contain conserved residues in RdRPs across all viral families. Motifs D, E, and G differ to various extents in sequence, in particular between the primer-dependent and the de novo RdRPs, but their spatial arrangements around the polymerase active site remain
The interplay between the RdRP and MTase modules in flaviviruses
Although viral RdRPs have been found to be naturally fused to capping enzymes in other viral families (e.g. the Rhabdoviridae and Paramyxoviridae families) (Li et al., 2008, Ogino et al., 2005, Ogino et al., 2010), the simple MTase-RdRP combination in flavivirus NS5 is very unique, thus being an attractive system to dissect how these two distinct enzymes coexist in a single poly-peptide. The NS5 MTase crystal structure was first solved in 2002, revealing a typical S-adenosyl-l-methionine
The conformational dynamics of flavivirus RdRP module
To date, crystal structures of RdRP module from WNV, DENV, and JEV, full-length crystal structures from JEV and DENV, and some RdRP containing structures in complex with NTP/NTP analogs or with small molecule inhibitors have been reported (Egloff et al., 2007, Klema et al., 2016, Lu and Gong, 2013, Noble et al., 2013, Surana et al., 2014, Tarantino et al., 2016, Yap et al., 2007, Yokokawa et al., 2016, Zhao et al., 2015). The modulation of RdRP by the MTase or ligands/inhibitors results in
Key missing links in structural aspect of flavivirus RdRPs
Among the remaining challenges with respect to the structure of flavivirus RdRP, the crystal structures of catalytic complexes are on the top of the list, to reveal the mechanisms of de novo initiation, the transition from initiation to elongation, and elongation. In particular, how the flavivirus RdRP priming element fulfills its function at initiation and what is its fate at elongation are of significant interest. The conformational diversity of full-length NS5 is another important aspect and
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported in part by the National Natural Science Foundation of China (31370198), the National Key Basic Research Program of China (2013CB911100), and the National Key Research and Development Program of China (2016YFC1200400).
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