NS3 helicase from dengue virus specifically recognizes viral RNA sequence to ensure optimal replication

Swarbrick, C.M.D.; Basavannacharya, Chandrakala; Chan, Kitti Wing Ki; Chan, Shu-Ann; Singh, Daljit; Wei, Na; Phoo, Wint Wint; Luo, Dahai; Lescar, Julien; Vasudevan, Subhash G.

doi:10.1093/nar/gkx1127

Cited by 63 publications

(61 citation statements)

References 71 publications

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“…Furthermore, it has been shown that some single-amino-acid mutation in R538, R225 and R268 of DENV, which affect the replication of DENV (17, 58). Another study report that the Asp-285-to-Ala substitution of the JEV NS3 protein abolished the ATPase and RNA helicase activities (63).…”

Section: Discussionmentioning

confidence: 99%

Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Jiang

Zhao

Bai

et al. 2019

Preprint

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27Previous studies revealed that Japanese encephalitis virus (JEV) infection alters the 28 expression of miRNA in central nervous system (CNS). However, the mechanism of JEV 29 infection contributes to the regulation of miRNAs in CNS remain obscure. Here, we found 30 that a global degradation of mature miRNA in mouse brain and neuroblastoma cells after JEV 31 infection. In additional, the integrative analysis of miRNAs and mRNAs suggests that those 32 down-regulated miRNAs are primarily targeted inflammation genes and the miR-466d-3p 33 target the IL-1β which in the middle of those inflammation genes. Transfection of 34 miR-466d-3p decreased the IL-1β expression and inhibited the JEV replication in NA cells. 35 Interestingly, the miR-466d-3p level increased after JEV infection in the presence of 36 cycloheximide, which indicated that viral protein expression reduces miR-466d-3p. 37Therefore, we generated all the JEV coding protein and demonstrated that NS3 is a potent 38 miRNA suppressor. Furthermore, the NS3 of ZIKA virus, WNV, DENV1 and DENV2 also 39 decreased the expression of miR-466d-3p. The in vitro unwinding assay demonstrated that the 40 NS3 could unwind the pre-miR-466d and induce the disfunction of miRNA. Using 41 computational models and RNA immunoprecipitation assay, we found that arginine-rich 42 domains of NS3 are critical for pre-miRNA binding and the degradation of host miRNAs. 43Importantly, site-directed mutagenesis of conserved residues revealed that R226G and 44 R202W significantly reduced the binding affinity and degradation of pre-miR-466d. 45 Together, these results extend the helicase of Flavivirus function beyond unwinding duplex 46 RNA to the decay of pre-miRNAs, which provides a new mechanism of NS3 in regulating 47 miRNA pathways and promoting the neuroinflammation. 48 49 Author Summary 50 Host miRNAs had been reported to regulate JEV induced inflammation in central nervous 51 system. We found that the NS3 of JEV can reduce most of host miRNA expression. The 52 helicase region of the NS3 specifically binds to precursors of miRNA and lead to incorrect 53 unwinding of precursors of miRNAs which inhibits the function of miRNAs. This observation 54 leads to two major findings. First, we identified the miR-466d-3p targets to the host IL-1β and 55 E protein of JEV, and NS3 degrades the miR-466d-3p to promote the brain inflammation and 56 viral replication. Second, we proved that the arginine on the helicase of NS3 is the main 57 miRNA binding sites, and the miRNA degradation by NS3 was abolished when the R226 and 58 R202 were mutated on the NS3. These findings were also confirmed with NS3 of ZIKA virus, 59WNV and DENV which could decrease the expression level of miR-466d-3p to enhance the 60 inflammation. Our study provides new insights into the molecular mechanism of encephalitis 61 caused by JEV, and reveals several amino acid sites to further attenuate the JEV vaccine. 62 63 65 JEV is a single-stranded, positive-sense RNA virus belonging to flavivirus of the Flaviviridae 66 family...

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Section: Discussionmentioning

confidence: 99%

Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Jiang

Zhao

Bai

et al. 2019

Preprint

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“…All remaining top conserved NS3 epitopes were found to lie within the conserved flavivirus motifs and map to the helicase domain [57]. Remarkably, while these NS3 epitopes were located far from each other in the primary structure ( Fig 4B), they localized in the tertiary structure around the groove that is important for interacting with viral RNA and facilitating its unwinding [50,58].…”

Section: (S5 Fig)mentioning

confidence: 97%

“…Most of the residues within these epitopes are located in regions that provide stability to the polymerase complex [52]. (B) (Left panel) Coverage of the top conserved NS3 epitopes (Fig 3) along the protein sequence, and (right panel) their locations in the corresponding tertiary structure (PDB ID 5XC6) [50]. Although these epitopes are spread out in the primary structure, they localize near the RNA binding groove of the helicase domain [58].…”

Section: Identifying Epitopes That Can Serve As Targets For a Universmentioning

confidence: 99%

Cross-serotypically conserved epitope recommendations for a universal T cell-based dengue vaccine

et al. 2020

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Dengue virus (DENV)-associated disease is a growing threat to public health across the globe. Co-circulating as four different serotypes, DENV poses a unique challenge for vaccine design as immunity to one serotype predisposes a person to severe and potentially lethal disease upon infection from other serotypes. Recent experimental studies suggest that an effective vaccine against DENV should elicit a strong T cell response against all serotypes, which could be achieved by directing T cell responses toward cross-serotypically conserved epitopes while avoiding serotype-specific ones. Here, we used experimentallydetermined DENV T cell epitopes and patient-derived DENV sequences to assess the cross-serotypic variability of the epitopes. We reveal a distinct near-binary pattern of epitope conservation across serotypes for a large number of DENV epitopes. Based on the conservation profile, we identify a set of 55 epitopes that are highly conserved in at least 3 serotypes. Most of the highly conserved epitopes lie in functionally important regions of DENV non-structural proteins. By considering the global distribution of human leukocyte antigen (HLA) alleles associated with these DENV epitopes, we identify a potentially robust subset of HLA class I and class II restricted epitopes that can serve as targets for a universal T cellbased vaccine against DENV while covering~99% of the global population.

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“…Interestingly, trans complementation with the NS3 mutants S138A or R461Q in which the protease and the helicase are inactive, respectively, rescued virus production, suggesting that the function of NS3 during virus assembly is independent from its known enzymatic activity. Lastly, structural studies suggest that binding of NS3 to specific 5′ UTR sequences could function as a molecular signature to guide newly synthesized vRNA out of the replication complex [99]. Nevertheless, biophysical and biochemical evidence to support this hypothesis is lacking.…”

Section: Post-replicative Functions Of Ns Proteinsmentioning

confidence: 99%

Role of RNA-binding proteins during the late stages of Flavivirus replication cycle

Diosa-Toro

Prasanth

Bradrick

et al. 2020

Virol J

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The genus Flavivirus encompasses several worldwide-distributed arthropod-borne viruses including, dengue virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, Zika virus, and tick-borne encephalitis virus. Infection with these viruses manifest with symptoms ranging from febrile illness to life-threatening hypotensive shock and encephalitis. Therefore, flaviviruses pose a great risk to public health. Currently, preventive measures are falling short to control epidemics and there are no antivirals against any Flavivirus. Flaviviruses carry a single stranded positive-sense RNA genome that plays multiple roles in infected cells: it is translated into viral proteins, used as template for genome replication, it is the precursor of the subgenomic flaviviral RNA and it is assembled into new virions. Furthermore, viral RNA genomes are also packaged into extracellular vesicles, e.g. exosomes, which represent an alternate mode of virus dissemination. Because RNA molecules are at the center of Flavivirus replication cycle, viral and host RNA-binding proteins (RBPs) are critical determinants of infection. Numerous studies have revealed the function of RBPs during Flavivirus infection, particularly at the level of RNA translation and replication. These proteins, however, are also critical participants at the late stages of the replication cycle. Here we revise the function of host RBPs and the viral proteins capsid, NS2A and NS3, during the packaging of viral RNA and the assembly of new virus particles. Furthermore, we go through the evidence pointing towards the importance of host RBPs in mediating cellular RNA export with the idea that the biogenesis of exosomes harboring Flavivirus RNA would follow an analogous pathway.

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NS3 helicase from dengue virus specifically recognizes viral RNA sequence to ensure optimal replication

Cited by 63 publications

References 71 publications

Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Cross-serotypically conserved epitope recommendations for a universal T cell-based dengue vaccine

Role of RNA-binding proteins during the late stages of Flavivirus replication cycle

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