Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV A-D), and the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and Coxsackievirus-A16 (CVA16) is a threat to global public health. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and possesses the RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing the anti-enteroviral drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein, and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, and both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. Besides, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, exerted effective antiviral activity against two important EV-Bs, Coxsackievirus B3 and Echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C by rationally designed peptide is an efficient antiviral strategy against enteroviruses, and the 2CL and B-2CL showed promising clinical potentials to be further developed as broad-spectrum anti-enteroviral drugs. Importance Enteroviruses are a large group of positive-sense single-stranded RNA viruses, and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved antiviral drug is available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71-and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo. Furthermore, we demonstrated that the strategy of targeting the 2C helicase activity can be used to other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antiviral for enteroviruses.

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Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.03216-15 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5384-5398 ◽

Cited By ~ 24

Author(s):

Long Liu ◽

Jiao Tian ◽

Hao Nan ◽

Mengmeng Tian ◽

Yuan Li ◽

...

Keyword(s):

Rna Synthesis ◽

Nonstructural Protein ◽

Critical Role ◽

Continuous Extension ◽

Rna Helicase ◽

Viral Rna ◽

Respiratory Syndrome Virus ◽

Nascent Transcript ◽

Multifunctional Protein ◽

N Protein

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.IMPORTANCEIt is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

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Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

Journal of Virology ◽

10.1128/jvi.03213-14 ◽

2014 ◽

Vol 89 (6) ◽

pp. 3145-3162 ◽

Cited By ~ 37

Author(s):

Age Utt ◽

Pratyush Kumar Das ◽

Margus Varjak ◽

Valeria Lulla ◽

Aleksei Lulla ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Chikungunya Virus ◽

Nonstructural Protein ◽

Rna Helicase ◽

Enzymatic Activities ◽

Host Factors ◽

Adaptive Mutations ◽

Nonstructural Protein 2 ◽

Experimental Systems

ABSTRACTChikungunya virus (CHIKV) (genusAlphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels.IMPORTANCECHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.

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Nucleoside Triphosphatase and RNA Helicase Activities Associated with GB Virus B Nonstructural Protein 3

Virology ◽

10.1006/viro.1999.9871 ◽

1999 ◽

Vol 261 (2) ◽

pp. 216-226 ◽

Cited By ~ 15

Author(s):

Weidong Zhong ◽

Paul Ingravallo ◽

Jacquelyn Wright-Minogue ◽

Angela Skelton ◽

Annette S. Uss ◽

...

Keyword(s):

Nonstructural Protein ◽

Rna Helicase ◽

Nonstructural Protein 3 ◽

Nucleoside Triphosphatase

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Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900656116 ◽

2019 ◽

Vol 116 (19) ◽

pp. 9558-9567 ◽

Cited By ~ 11

Author(s):

Yee-Song Law ◽

Age Utt ◽

Yaw Bia Tan ◽

Jie Zheng ◽

Sainan Wang ◽

...

Keyword(s):

Atpase Activity ◽

Chikungunya Virus ◽

Rna Binding ◽

Nonstructural Protein ◽

Rna Helicase ◽

Viral Rna ◽

Helicase Domain ◽

Stacking Interactions ◽

Adaptive Mutations ◽

Rna Backbone

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3′-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transition-state nucleotide analog ADP-AlF4. Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.

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Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Journal of Virology ◽

10.1128/jvi.75.17.8289-8297.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8289-8297 ◽

Cited By ~ 46

Author(s):

Chun-Ling Tai ◽

Wen-Ching Pan ◽

Shwu-Huey Liaw ◽

Ueng-Cheng Yang ◽

Lih-Hwa Hwang ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Atpase Activity ◽

Rna Binding ◽

Mutational Analysis ◽

Nonstructural Protein ◽

Rna Helicase ◽

Helicase Domain ◽

Helicase Activity ◽

Conserved Sequence

ABSTRACT The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

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The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB

Virology ◽

10.1016/j.virol.2014.07.037 ◽

2014 ◽

Vol 464-465 ◽

pp. 353-364 ◽

Cited By ~ 3

Author(s):

Fenfen Tang ◽

Hongjie Xia ◽

Peipei Wang ◽

Jie Yang ◽

Tianyong Zhao ◽

...

Keyword(s):

Nucleic Acid ◽

Nonstructural Protein ◽

Enterovirus 71 ◽

Chaperone Activity ◽

Human Enterovirus ◽

Human Enterovirus 71 ◽

Nucleic Acid Chaperone ◽

Identification And Characterization

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Structural and Functional Studies of Chikungunya Virus nsP2

Proceedings ◽

10.3390/proceedings2020050113 ◽

2020 ◽

Vol 50 (1) ◽

pp. 113

Author(s):

Yee Song Law ◽

Yaw Bia Tan ◽

Orion Shih ◽

Age Utt ◽

Jie Zheng ◽

...

Keyword(s):

Cysteine Protease ◽

Solution Structure ◽

Chikungunya Virus ◽

Atp Hydrolysis ◽

Nonstructural Protein ◽

Rna Helicase ◽

Full Length ◽

Viral Rna ◽

Helicase Domain ◽

Functional Studies

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) contains an N-terminal RNA helicase with both nucleotide triphosphatase and RNA triphosphatase activities, and a C-terminal cysteine protease that is responsible for polyprotein processing. Both N-terminal RNA helicase and C-terminal cysteine protease are connected through a flexible linker. Although the structure of the C-terminal cysteine protease has been solved, the structure and the conformational arrangement of full-length nsP2 remains elusive. Here, we determined the crystal structure of the helicase part of the CHIKV nsP2 (nsP2h) bound to the conserved 3′-end of the genomic RNA and the nucleotide analogue ADP-AlF4. The structure of this ternary complex revealed the molecular basis for viral RNA recognition and ATP hydrolysis by the nsP2h. Unique hydrophobic protein–RNA interactions play essential roles in viral RNA replication. We also determined the solution structure of full-length nsP2 using small-angle X-ray scattering (SAXS). The solution architecture of the nsP2 was modeled using the available high-resolution structures and program CORAL (complexes with random loops). The CORAL model revealed that nsP2 is partially unfolded and the N-terminal protease domain is arranged near the N-terminal domain of the helicase domain. These findings expand our knowledge of CHIKV and related alphaviruses and might also have broad implications for antiviral and vaccine developments against pathogenic alphaviruses.

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The Cellular RNA Helicase DDX1 Interacts with Coronavirus Nonstructural Protein 14 and Enhances Viral Replication

Journal of Virology ◽

10.1128/jvi.00392-10 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8571-8583 ◽

Cited By ~ 56

Author(s):

Linghui Xu ◽

Siti Khadijah ◽

Shouguo Fang ◽

Li Wang ◽

Felicia P. L. Tay ◽

...

Keyword(s):

Nonstructural Protein ◽

Rna Helicase ◽

Host Proteins ◽

Bait Protein ◽

Infectious Bronchitis ◽

Infected Cells ◽

Interfering Rna ◽

Discontinuous Transcription ◽

Two Hybrid ◽

Hybrid Screening

ABSTRACT The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.

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Rapid Degradation of Hfq-Free RyhB inYersinia pestisby PNPase Independent of Putative Ribonucleolytic Complexes

BioMed Research International ◽

10.1155/2014/798918 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Zhongliang Deng ◽

Zizhong Liu ◽

Yujing Bi ◽

Xiaoyi Wang ◽

Dongsheng Zhou ◽

...

Keyword(s):

Rna Helicase ◽

Binding Domain ◽

Rnase E ◽

Rnase Iii ◽

Rna Chaperone ◽

Rapid Degradation ◽

Transcript Levels

The RNA chaperone Hfq in bacteria stabilizes sRNAs by protecting them from the attack of ribonucleases. Upon release from Hfq, sRNAs are preferably degraded by PNPase. PNPase usually forms multienzyme ribonucleolytic complexes with endoribonuclease E and/or RNA helicase RhlB to facilitate the degradation of the structured RNA. However, whether PNPase activity on Hfq-free sRNAs is associated with the assembly of RNase E or RhlB has yet to be determined. Here we examined the roles of the main endoribonucleases, exoribonucleases, and ancillary RNA-modifying enzymes in the degradation ofY. pestisRyhB in the absence of Hfq. Expectedly, the transcript levels of both RyhB1 and RyhB2 increase only after inactivating PNPase, which confirms the importance of PNPase in sRNA degradation. By contrast, the signal of RyhB becomes barely perceptible after inactivating of RNase III, which may be explained by the increase in PNPase levels resulting from the exemption ofpnpmRNA from RNase III processing. No significant changes are observed in RyhB stability after deletion of either the PNPase-binding domain of RNase E orrhlB. Therefore, PNPase acts as a major enzyme of RyhB degradation independent of PNPase-containing RNase E and RhlB assembly in the absence of Hfq.

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