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Review
. 2021 May 21;13(6):956.
doi: 10.3390/v13060956.

Molecular Insights into the Flavivirus Replication Complex

Kaïn van den Elsen  1   2   3 , Jun Ping Quek  1   2 , Dahai Luo  1   2   4
Affiliations
Review

Molecular Insights into the Flavivirus Replication Complex

Kaïn van den Elsen et al. Viruses. .

Abstract

Flaviviruses are vector-borne RNA viruses, many of which are clinically relevant human viral pathogens, such as dengue, Zika, Japanese encephalitis, West Nile and yellow fever viruses. Millions of people are infected with these viruses around the world each year. Vaccines are only available for some members of this large virus family, and there are no effective antiviral drugs to treat flavivirus infections. The unmet need for vaccines and therapies against these flaviviral infections drives research towards a better understanding of the epidemiology, biology and immunology of flaviviruses. In this review, we discuss the basic biology of the flavivirus replication process and focus on the molecular aspects of viral genome replication. Within the virus-induced intracellular membranous compartments, flaviviral RNA genome replication takes place, starting from viral poly protein expression and processing to the assembly of the virus RNA replication complex, followed by the delivery of the progeny viral RNA to the viral particle assembly sites. We attempt to update the latest understanding of the key molecular events during this process and highlight knowledge gaps for future studies.

Keywords: RNA replication; flavivirus; non-structural protein; replication complex.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 4
Figure 4
Relationship between different conformational states of the Flavivirus full-length NS3 structures. The protein structures were obtained from PDB and adapted from [16,86]. Conformation 1 (PDB: 2VBC) and conformation 2 (PDB: 2WHX) are shown. Ribbon representation showing the arrangement of the protease and helicase domains of NS3. The NS2B cofactor is coloured in magenta, protease domain is in yellow and the helicase domain in green. The conformational changes between the protein structures were analysed using the DynDom database [93].
Figure 5
Figure 5
Cartoon representation of the flavivirus capping process. During the RNA capping process, GTP binds to the GTP binding pocket of NS5 and forms an NS5–GMP intermediate. Concurrently, NS3 RTPase removes phosphate from the viral genome to form a diphosphorylated RNA product. The NS5–GMP intermediate subsequently transfers the GMP to the diphosphorylated RNA upon RNA binding. Then, GpppA-RNA reposition the guanosine near the SAM binding site for N7-methylation. The SAH molecule is replenished by a SAM molecule while the N7MGpppA-RNA undergoes another repositioning to allow the initiating adenosine to be positioned near the SAM binding site for 2′O methylation to generate N7MGpppA2′OM-RNA. Representative protein structures with their PDB codes are also shown.
Figure 6
Figure 6
Relationship between the different conformational states of the Flavivirus full-length NS5 structures. The protein structures were obtained from PDB and adapted from [115,118,119,121]. JEV (PDB: 4K6M), YFV (PDB: 6QSN), DENV2 (PDB: 6KR2), DENV2 (PDB: 6KR3) and DENV3 (PDB: 4V0Q). Ribbon representations show the arrangement of the MT and RdRp domains of NS5. The MTase is coloured in cyan, the RdRp fingers in green, the RdRp palm in brown and the RdRp thumb in blue. The conformational changes between the protein structures were analysed using the DynDom database [93].
Figure 1
Figure 1
The flavivirus replication cycle. (A) Cartoon representation of the steps in the replication cycle, as detailed earlier in the section. (B) The translation and RNA replication step of replication are enlarged, showing the viral polyprotein, possible arrangement of the proteins in the complex, and the virion packaging mechanism. These steps require further study to fully elucidate how these structures assemble and function [15,16,17,18,19,20,21]. Created with Biorender.com.
Figure 1
Figure 1
The flavivirus replication cycle. (A) Cartoon representation of the steps in the replication cycle, as detailed earlier in the section. (B) The translation and RNA replication step of replication are enlarged, showing the viral polyprotein, possible arrangement of the proteins in the complex, and the virion packaging mechanism. These steps require further study to fully elucidate how these structures assemble and function [15,16,17,18,19,20,21]. Created with Biorender.com.
Figure 2
Figure 2
Flavivirus replication organelle. A representation of a possible configuration of the replication complex based on current understanding. Further study is required to elucidate how the interplay between these, the ROs and the host influences viral propagation [15,16,17]. Created with Biorender.com.
Figure 3
Figure 3
Cartoon representation of the flavivirus polyprotein. The cleavage site of viral protease is represented by a solid arrow, while the cleavage site by host protease is represented by an open arrow. A summary of the function of the viral proteins is described.
Figure 7
Figure 7
Schematic representation of the flavivirus genome. The figure is adapted from [145]. The viral genome is capped with a single open reading frame that is flanked by both 5′ and 3′ UTRs. Functional RNA elements in the 5′ UTR include stem-loop (SL) A, SLB, the upstream AUG region (UAR), UAR-flanking stem (UFS) and the capsid-coding region hairpin (cHP). The 3′ UTR can be classified into three regions based on sequence conservation and includes functional elements such as DB and 3′SL. These secondary structures and functional RNA elements are shown and annotated. The viral genome is also proposed to undergo genome cyclisation. The circularisation is mediated by sets of inverted complementary sequences 5′UAR-3′UAR, and 5′ downstream AUG region (DAR)-3′DAR, 5′ cyclisation sequence (CS)-3′CS, as illustrated. The predicted subgenomic RNAs sfRNA1 and sfRNA2 are also formed depending on the location in which Xrn-1 is installed.
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