Supramolecular arrangement of the full-length Zika virus NS5

Ferrero, D.S.; Ruiz-Arroyo, Victor M.; Soler, Nicolas; Usón, Isabel; Guarné, Alba; Verdaguer, N.

doi:10.1371/journal.ppat.1007656

Cited by 45 publications

(51 citation statements)

References 69 publications

(85 reference statements)

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“…Full-length NS5 showed a low Hill coefficient and was not complemented by NS5-GNN. Thus, our results support the idea that USUV is similar to DENV and Zika virus (ZIKV) polymerases, and previous structural data suggest that their NS5 polymerases form homodimers (25,28,(39)(40)(41).…”

Section: Discussionsupporting

confidence: 89%

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Albentosa-González¹,

Clemente-Casares

Sabariegos

et al. 2019

Antimicrob Agents Chemother

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Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyltransferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we cloned and expressed the entire NS5 and the RdRpD in a heterologous system and used purified preparations for protein characterizations. We determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA and that the RdRp activity is not inhibited by sofosbuvir. To further examine the characteristics of USUV polymerase in a more specifically biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed their subcellular location. NS5 is predominantly found in the cytoplasm; a significant proportion is directed to the nucleus, and this translocation involves nuclear location signals (NLS) located at least between the MTase and RdRpD domains.

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Section: Discussionsupporting

confidence: 89%

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Albentosa-González¹,

Clemente-Casares

Sabariegos

et al. 2019

Antimicrob Agents Chemother

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“…To date, two types of global conformations have been observed in full-length NS5 structures [11]. The conformation revealed by the JEV NS5 structure (named JEV-mode hereinafter) features a medium size interface (~1540 Å 2 , for all interface area values presented in this study, the total buried solvent accessible surface from both side of the interface was accounted) with a conserved hydrophobic core [8,12], and is also observed in recently reported ZIKV, yellow fever virus (YFV), and DENV serotype 2 (DENV2) full-length NS5 crystal structures [13][14][15][16][17][18]. In such a conformation, the MTase approaches the RdRP from its backside and interacts with the RdRP middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal (NLS-helix) [19] ( Fig 1A).…”

Section: Introductionsupporting

confidence: 67%

“…With two monomer conformation modes and eight crystal forms identified, more than 10 NS5 dimer interfaces can be recognized in the aforementioned NS5 crystal structures with no obvious conservative features. A couple of studies did focus on some of these dimer interface interactions, even though the primary NS5 solution state is monomer [16,21]. Either by probing the inter-molecular interactions, deleting the MTase domain, or mutating the MTase-RdRP linker, multiple in vitro polymerase assay-based studies together suggest that the MTase regulates the RdRP catalytic activities, albeit to overall moderate extents [12,14,16,18,24,25].…”

Section: Plos Pathogensmentioning

confidence: 99%

A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase

Zhang

et al. 2020

PLoS Pathog

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The flaviviruses pose serious threats to human health. Being a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP), NS5 is the most conserved flavivirus protein and an important antiviral target. Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. However, whether these NS5 conformations are conserved in flaviviruses and their specific functions remain elusive. Here we report two forms of DENV serotype 2 (DENV2) NS5 crystal structures representing two conformational states with defined analogies to the JEV-mode and DENV3-mode conformations, respectively, demonstrating the conservation of both conformation modes and providing clues for how different conformational states may be interconnected. Data from in vitro polymerase assays further demonstrate that perturbing the JEV-mode but not the DENV3-mode intramolecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. Our work highlights the role of MTase as a unique intra-molecular initiation factor specifically only through the JEV-mode conformation, providing an example of conformation-based crosstalk between naturally fused protein functional modules.

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“…Findings from the interactome prompted us to study the subcellular distribution of ZikV-NS5 in NPCs by introducing a plasmid encoding ZikV-NS5-FLAG tagged into the chick embryo NT. As expected given the nuclear localization signals encoded in this protein, the NS5-FLAG protein accumulated in the nucleus 12 (Figs. 1f-h), a subcellular distribution unique among the ZikV proteins (Extended data Figs.…”

Section: Resultssupporting

confidence: 80%

“…ZikV-NS5 is the largest NS protein (~100 kDa) and it is essential to the viral life cycle. It contains two functional domains, a N-terminal methyltransferase (MTase) domain responsible for the catalysis of 5’-mRNA capping and methylation, and a RNA-dependent RNA polymerase (RdRP) domain at the C-terminus that drives genome replication 12–15 . ZikV-NS5 can form dimers and higher-order oligomers that regulate its enzymatic functions, as well as its association with other viral or host factors during infection, making it an extremely attractive anti-viral target 13 .…”

Section: Introductionmentioning

confidence: 99%

Multimerization of Zika Virus-NS5 causes a ciliopathy and forces premature neurogenesis

Saade

Ferrero

Blanco-Ameijeiras

et al. 2019

Preprint

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33Zika virus (ZikV) is a flavivirus that infects neural tissues, causing congenital 34 microcephaly. ZikV has evolved multiple mechanisms to restrict proliferation and 35 enhance cell death, although the underlying cellular events involved remain unclear. 36 Here we show that the ZikV-NS5 protein interacts with host proteins at the base of the 37 primary cilia in neural progenitor cells, causing an atypical non-genetic ciliopathy and 38 premature neuron delamination. Furthermore, in human microcephalic fetal brain 39 tissue, ZikV-NS5 persists at the base of the motile cilia in ependymal cells, which also 40 exhibit a severe ciliopathy. While the enzymatic activity of ZikV-NS5 appears to be 41 dispensable, the Y25, K28 and K29 residues in the protein, that are involved in NS5-42 oligomerization, are essential for the localization and interaction with components of the 43 cilium base, promoting ciliopathy and premature neurogenesis. These findings lay the 44 foundation to develop therapies that target ZikV-NS5-multimerization, preventing the 45 developmental malformations associated with congenital Zika syndrome 46 47 48 Zika virus (ZikV) is a flavivirus transmitted by the bite of the Aedes mosquito 1 . The 11 49 kb positive-sense, single-stranded RNA genome of ZikV encodes ten mature viral 50 proteins: three structural proteins (C, prM and E) and seven non-structural proteins 51 (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) 1 . A single amino acid substitution 52 (S139N) in prM seems to be responsible for the change in virus tropism that provokes a 53 dramatic increase in neonatal microcephaly 22 , and that has led to this virus being 54 declared a global threat to public health. ZikV can directly infect human neural 55 progenitor cells (NPCs) in both 2D and 3D in vitro models of the developing cerebral 56 cortex, resulting in defects resembling congenital microcephaly 3-8 . However, the 57 mechanisms by which ZikV disrupts neurogenesis have not yet been fully elucidated. 58Moreover, macaque monkey infection provokes regional disturbances in the brain that 59 impair postnatal neurogenesis, provoking cognitive deficits and epilepsy 9 . This 60 vulnerability of late neurogenic regions to ZikV also persists in adult mice 10 . As it is 61 well known that human brain development extends for many years beyond birth and 62 that adult neurogenesis influences cognitive functions, it is no longer safe to consider 63 ZikV as a transient infection in adult humans without marked long-term effects. 64 65 The limited availability of human tissue to perform histological analyses at different 66 developmental stages 11 emphasizes the need to use in vivo animal models to understand 67 congenital Zika syndrome. Here we used the chick embryo neural tube (NT) to screen 68 non-structural (NS) ZikV protein components and assess their impact on neurogenesis, 69 particularly given that this in vivo model has served to identify fundamental processes in 70 mammalian neural development and human disease. Through this approach, ZikV-NS5 71 w...

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Supramolecular arrangement of the full-length Zika virus NS5

Cited by 45 publications

References 69 publications

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase

Multimerization of Zika Virus-NS5 causes a ciliopathy and forces premature neurogenesis

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