Interactions Between the Dengue Virus Polymerase NS5 and Stem-Loop A

Overview

Journal J Virol

Publisher American Society for Microbiology

Date 2017 Mar 31

PMID 28356528

Citations 22

Authors

Paul J Bujalowski

Wlodzimierz Bujalowski

Kyung H Choi

Affiliations

Soon will be listed here.

Abstract

The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5' untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA and SLA, which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides. Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5' untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the energetics of intrinsic affinities, characterize the stoichiometry of the complex of NS5 and SLA, and determine how solution conditions such as magnesium and sodium concentrations and temperature influence NS5-SLA interactions in solution. Quantitatively characterizing dengue virus NS5-SLA interactions will facilitate the design and assessment of antiviral therapeutics that target this essential step of the dengue virus life cycle.

Citing Articles

Structural Dynamics of the Dengue Virus Non-structural 5 (NS5) Interactions with Promoter Stem Loop A (SLA).

Obi J, Kihn K, McQueen L, Fields J, Snyder G, Deredge D bioRxiv. 2024; .

PMID: 39677779 PMC: 11642867. DOI: 10.1101/2024.12.03.626708.

Dengue virus: pathogenesis and potential for small molecule inhibitors.

Chauhan N, Gaur K, Asuru T, Guchhait P Biosci Rep. 2024; 44(8).

PMID: 39051974 PMC: 11327219. DOI: 10.1042/BSR20240134.

The myriad roles of RNA structure in the flavivirus life cycle.

Abram Q, Landry B, Wang A, Kothe R, Hauch H, Sagan S RNA Biol. 2024; 21(1):14-30.

PMID: 38797925 PMC: 11135854. DOI: 10.1080/15476286.2024.2357857.

High-resolution RNA tertiary structures in Zika virus stem-loop A for the development of inhibitory small molecules.

Tipo J, Gottipati K, Choi K RNA. 2024; 30(6):609-623.

PMID: 38383158 PMC: 11098461. DOI: 10.1261/rna.079796.123.

Binding of microRNA-122 to the hepatitis C virus 5' untranslated region modifies interactions with poly(C) binding protein 2 and the NS5B viral polymerase.

Scott S, Li Y, Bermek O, Griffith J, Lemon S, Choi K Nucleic Acids Res. 2023; 51(22):12397-12413.

PMID: 37941151 PMC: 10711565. DOI: 10.1093/nar/gkad1000.

References

Lodeiro M, Filomatori C, Gamarnik A . Structural and functional studies of the promoter element for dengue virus RNA replication. J Virol. 2008; 83(2):993-1008. PMC: 2612346. DOI: 10.1128/JVI.01647-08. View

Filomatori C, Iglesias N, Villordo S, Alvarez D, Gamarnik A . RNA sequences and structures required for the recruitment and activity of the dengue virus polymerase. J Biol Chem. 2010; 286(9):6929-39. PMC: 3044948. DOI: 10.1074/jbc.M110.162289. View

Villordo S, Carballeda J, Filomatori C, Gamarnik A . RNA Structure Duplications and Flavivirus Host Adaptation. Trends Microbiol. 2016; 24(4):270-283. PMC: 4808370. DOI: 10.1016/j.tim.2016.01.002. View

Issur M, Geiss B, Bougie I, Picard-Jean F, Despins S, Mayette J . The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure. RNA. 2009; 15(12):2340-50. PMC: 2779676. DOI: 10.1261/rna.1609709. View

Bujalowski W . Thermodynamic and kinetic methods of analyses of protein-nucleic acid interactions. From simpler to more complex systems. Chem Rev. 2006; 106(2):556-606. DOI: 10.1021/cr040462l. View

Saw W, Tria G, Gruber A, Subramanian Manimekalai M, Zhao Y, Chandramohan A . Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution. Acta Crystallogr D Biol Crystallogr. 2015; 71(Pt 11):2309-27. PMC: 4631481. DOI: 10.1107/S1399004715017721. View

Hammond J, Rambo R, Filbin M, Kieft J . Comparison and functional implications of the 3D architectures of viral tRNA-like structures. RNA. 2009; 15(2):294-307. PMC: 2648705. DOI: 10.1261/rna.1360709. View

Tolman G, Barrio J, LEONARD N . Chloroacetaldehyde-modified dinucleoside phosphates. Dynamic fluorescence quenching and quenching due to intramolecular complexation. Biochemistry. 1974; 13(24):4869-78. DOI: 10.1021/bi00721a001. View

You S, Padmanabhan R . A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA. J Biol Chem. 1999; 274(47):33714-22. DOI: 10.1074/jbc.274.47.33714. View

10.

Jezewska M, Bujalowski P, Bujalowski W . Interactions of the DNA polymerase X from African swine fever virus with gapped DNA substrates. Quantitative analysis of functional structures of the formed complexes. Biochemistry. 2007; 46(45):12909-24. DOI: 10.1021/bi700677j. View

11.

Guzman M, Kouri G . Dengue: an update. Lancet Infect Dis. 2002; 2(1):33-42. DOI: 10.1016/s1473-3099(01)00171-2. View

12.

Guzman M, Alvarez M, Rodriguez R, Rosario D, Vazquez S, Vald s L . Fatal dengue hemorrhagic fever in Cuba, 1997. Int J Infect Dis. 1999; 3(3):130-5. DOI: 10.1016/s1201-9712(99)90033-4. View

13.

EDELHOCH H . Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967; 6(7):1948-54. DOI: 10.1021/bi00859a010. View

14.

Potisopon S, Priet S, Collet A, Decroly E, Canard B, Selisko B . The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain. Nucleic Acids Res. 2014; 42(18):11642-56. PMC: 4191377. DOI: 10.1093/nar/gku666. View

15.

Bhatt S, Gething P, Brady O, Messina J, Farlow A, Moyes C . The global distribution and burden of dengue. Nature. 2013; 496(7446):504-7. PMC: 3651993. DOI: 10.1038/nature12060. View

16.

Bussetta C, Choi K . Dengue virus nonstructural protein 5 adopts multiple conformations in solution. Biochemistry. 2012; 51(30):5921-31. PMC: 3448003. DOI: 10.1021/bi300406n. View

17.

Choi K, Rossmann M . RNA-dependent RNA polymerases from Flaviviridae. Curr Opin Struct Biol. 2009; 19(6):746-51. DOI: 10.1016/j.sbi.2009.10.015. View

18.

Record Jr M, Lohman M, De Haseth P . Ion effects on ligand-nucleic acid interactions. J Mol Biol. 1976; 107(2):145-58. DOI: 10.1016/s0022-2836(76)80023-x. View

19.

Marcinowicz A, Jezewska M, Bujalowski P, Bujalowski W . Structure of the tertiary complex of the RepA hexameric helicase of plasmid RSF1010 with the ssDNA and nucleotide cofactors in solution. Biochemistry. 2007; 46(46):13279-96. DOI: 10.1021/bi700729k. View

20.

Hodge K, Tunghirun C, Kamkaew M, Limjindaporn T, Yenchitsomanus P, Chimnaronk S . Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication. J Biol Chem. 2016; 291(33):17437-49. PMC: 5016140. DOI: 10.1074/jbc.M116.724013. View