Structure and biochemical analysis of Kokobera virus helicase

Speroni, Silvia; Colibus, Luigi De; Mastrangelo, Eloise; Gould, Ernest A.; Coutard, Bruno; Forrester, Naomi L.; Blanc, Séverine; Canard, Bruno; Mattevi, Andrea

doi:10.1002/prot.21812

Cited by 12 publications

(12 citation statements)

References 15 publications

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“…In addition, two conserved Arg residues in domain II (Arg458 and Arg461: “arginine fingers” in motif VI) are involved in ATPase and RTPase activities (Sampath et al, 2006), and in the structural rearrangement that results in RNA unwinding, following ATP hydrolysis. A similar role in coupling the ATPase and RNA unwinding activities is played by the residues of the Ia motif, as gathered from mutagenesis studies on the KOKV NS3Hel (Speroni et al, 2008). The central region of the NS3Hel, where the three domains contact each other, hosts a cleft held to be involved in ssRNA binding during the helicase activity (Xu et al, 2005; Luo et al, 2008b).…”

Section: Structure and Function Of Flaviviral Enzymesmentioning

confidence: 88%

“…Crystal structures of the flavivirus C-terminal NS3RTPase/Hel domain have been solved for YFV (Wu et al, 2005), DENV (Xu et al, 2005; Luo et al, 2008b) and JEV (Yamashita et al, 2008). In the context of the VIZIER Project, three new structures have been obtained for MVEV (Mancini et al, 2007); 1.9 Å resolution), KUNV, an Australian variant of WNV (Mastrangelo et al, 2007b; 3.1 Å) and KOKV (Speroni et al, 2008; 2.1 Å). In particular, the KOKV NS3Hel domain features high thermostability and good propensity to crystallize, making this an attractive model system for structural and biochemical analysis of inhibitor binding.…”

Section: Structure and Function Of Flaviviral Enzymesmentioning

confidence: 99%

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Structure and functionality in flavivirus NS-proteins: Perspectives for drug design

et al. 2010

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Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

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Section: Structure and Function Of Flaviviral Enzymesmentioning

confidence: 88%

Section: Structure and Function Of Flaviviral Enzymesmentioning

confidence: 99%

Structure and functionality in flavivirus NS-proteins: Perspectives for drug design

et al. 2010

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“…The flavivirus NS3 C-terminal NTPase/helicase has been solved for YFV (58), DENV (35,59), MVEV (38), KUNV (39), JEV (60), and Kokobera virus (49). It is a DEAH/D box helicase belonging to the SF2 superfamily of helicases containing the characteristic Walker A and B motifs responsible for NTP and Mg 2ϩ binding and consisting of three domains of approximately equal size, forming a large positively charged cleft at their juncture, which is the RNA binding site.…”

Section: Where I I (H) Is the Ith Measurement And ͻI(h)ͼ Is The Weighmentioning

confidence: 99%

“…The atomic structures of NS3pro in the presence and absence of ligands and/or the NS2B activating domain (2,19,47) and NS3hel (35,38,39,49,(58)(59)(60) are known, and recently, the structure of full-length DENV4 (one of four dengue virus serotypes) NS3 fused to an 18-residue NS2B cofactor (NS2B 18 NS3) was reported (34). This structure revealed an elongated conformation, with the protease domain interfacing with the NTP binding pocket and being separated from NS3hel by a relatively flexible linker, which suggested that the protease domain may have a positive effect on the activity of the NTPase/helicase domain.…”

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confidence: 99%

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

Assenberg

Mastrangelo

Walter

et al. 2009

J Virol

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122

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The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.Flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) belong to the family Flaviviridae and are the causative agents of a range of serious human diseases including hemorrhagic fever, meningitis, and encephalitis (37). They remain a global health priority, as many viruses are endemic in large parts of the Americas, Africa, Australia, and Asia, and vaccines remain unavailable for most members (31,46,57).Flaviviruses have a positive-sense single-stranded RNA (ssRNA) genome (approximately 11 kb) that encodes one large open reading frame containing a 5Ј type 1 cap and conserved RNA structures at both the 5Ј and 3Ј untranslated regions that are important for viral genome translation and replication. The genomic RNA is translated into a single polyprotein precursor (11) consisting of three structural (C [capsid], prM [membrane], and E [envelope]) and seven nonstructural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) proteins arranged in the order C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 (reviewed in reference 33) (Fig. 1). Only the structural proteins become part of the mature, infectious virion, whereas the nonstructural proteins are involved in polyprotein processing, viral RNA synthesis, and virus morphogenesis (33, 43). The precursor protein is directed by signal sequences into the host endoplasmic reticulum (ER), where NS1 and the exogenous domains of prM and E face the lumen, while C, ...

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“…2a). It is not uncommon for an NTPase to co-purify with a pyrophosphate, as for example, in structures of KOKV NS3-Hel 8 and H.pylori GTP cyclohydrolase 9 . A β-hairpin extends from domain 2 and interacts with domain 3 and has been proposed to act as a “wedge” to help separate the RNA strands of dsRNA for the unwinding reaction (Fig.…”

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confidence: 99%

Structure of the NS3 helicase from Zika virus

Jain

Coloma

García‐Sastre

et al. 2016

Nat Struct Mol Biol

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The Zika virus has emerged as a pathogen of major health concern. We present a high-resolution (1.62Å) crystal structure of the RNA helicase from the French Polynesia strain. The structure is similar to that of the RNA helicase from Dengue virus, with variability in the conformations of loops typically involved in ATP and RNA binding. We identify druggable “hotspots” that are well-suited for in-silico and/or fragment-based high throughput drug discovery.

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Structure and biochemical analysis of Kokobera virus helicase

Cited by 12 publications

References 15 publications

Structure and functionality in flavivirus NS-proteins: Perspectives for drug design

Structure and functionality in flavivirus NS-proteins: Perspectives for drug design

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

Structure of the NS3 helicase from Zika virus

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