Identification of an intramolecular switch that controls the interaction of the helicase nsp10 with the membrane-associated nsp12 of porcine reproductive and respiratory syndrome virus

A critical step in replication of positive-stranded RNA viruses is the assembly of replication and transcription complexes (RTC). We have recently mapped the nonstructural protein (nsp) interaction network of porcine reproductive and respiratory syndrome virus (PRRSV) and provided evidence by truncation mutagenesis that the recruitment of viral core replicase enzymes (nsp9 and nsp10) to membrane proteins (nsp2, nsp3, nsp5 and nsp12) is subject to regulation. Here, we went further to discover an intramolecular switch within the helicase nsp10 that controls its interaction with the membrane-associated protein nsp12. Deletion of nsp10 linker region aa.124-133 connecting the domain 1B to 1A led to complete re-localization and co-localization in the cells co-expressing nsp12. Moreover, single amino acid substitutions (e.g., nsp10 E131A and I132A) were sufficient to enable the nsp10-nsp12 interaction. Further proof came from membrane floatation assays that revealed a clear movement of nsp10 mutants, but not WT nsp10, towards the top of sucrose gradients in the presence of nsp12. Interestingly, the same mutations were not able to activate the nsp10-nsp2/3 interaction, suggesting a differential requirement for conformation. Reverse genetics analysis showed that PRRSV mutants carrying the single substitutions were not viable and defective of subgenomic RNA (sg RNA) accumulation. Together, our results provide strong evidence for a regulated interaction between nsp10 and nsp12 and suggest an essential role for an orchestrated RTC assembly in sg RNA synthesis. IMPORTANCE Assembly of replication and transcription complexes (RTC) is a limiting step for viral RNA synthesis. The PRRSV RTC macromolecular complexes are comprised of mainly viral nonstructural replicase proteins (nsps), but how they come together remains elusive. We previously showed that viral helicase nsp10 interacts nsp12 in a regulated manner by truncation mutagenesis. Here we revealed that the interaction is controlled by single residues within the domain linker region of nsp10. Moreover, the activation mutations leads to defect in viral sg RNA synthesis. Our results provide important insight into the mechanisms of PRRSV RTC assembly and regulation of viral sg RNA synthesis.

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Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

Journal of Virology ◽

10.1128/jvi.01112-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 2

Author(s):

Jiangwei Song ◽

Yuanyuan Liu ◽

Peng Gao ◽

Yunhao Hu ◽

Yue Chai ◽

...

Keyword(s):

Mammalian Cells ◽

Rna Virus ◽

Nonstructural Protein ◽

Transmembrane Proteins ◽

Interaction Network ◽

Host Cells ◽

Respiratory Syndrome Virus ◽

Reading Frame ◽

Interaction Map ◽

Transcription Complexes

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus belonging to the familyArteriviridae. Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are mainly composed of a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screening in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.IMPORTANCESynthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of RTC that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of the PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly.

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The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

Journal of Virology ◽

10.1128/jvi.00834-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9449-9463 ◽

Cited By ~ 58

Author(s):

Jun Han ◽

Mark S. Rutherford ◽

Kay S. Faaberg

Keyword(s):

Cysteine Protease ◽

Reverse Genetics ◽

Nonstructural Protein ◽

Point Mutations ◽

Respiratory Syndrome Virus ◽

Protease Domain ◽

Nonstructural Protein 2 ◽

Cleavage Activity ◽

Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

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Identification of Mutations Causing Temperature-Sensitive Defects in Semliki Forest Virus RNA Synthesis

Journal of Virology ◽

10.1128/jvi.80.6.3108-3111.2006 ◽

2006 ◽

Vol 80 (6) ◽

pp. 3108-3111 ◽

Cited By ~ 14

Author(s):

Valeria Lulla ◽

Andres Merits ◽

Peter Sarin ◽

Leevi Kääriäinen ◽

Sirkka Keränen ◽

...

Keyword(s):

Rna Synthesis ◽

Semliki Forest Virus ◽

Nonstructural Protein ◽

Prototype Strain ◽

Temperature Sensitive ◽

Helicase Domain ◽

Coding Region ◽

Protein Coding ◽

Subgenomic Rna ◽

The Individual

ABSTRACT We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.

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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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A Single Amino Acid in Nonstructural Protein NS4B Confers Virulence to Dengue Virus in AG129 Mice through Enhancement of Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.00665-11 ◽

2011 ◽

Vol 85 (15) ◽

pp. 7775-7787 ◽

Cited By ~ 61

Author(s):

D. Grant ◽

G. K. Tan ◽

M. Qing ◽

J. K. W. Ng ◽

A. Yip ◽

...

Keyword(s):

Amino Acid ◽

Dengue Virus ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Viral Rna ◽

Single Amino Acid

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The nsp2 Hypervariable Region of Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06 Is Associated with Viral Cellular Tropism to Primary Porcine Alveolar Macrophages

Journal of Virology ◽

10.1128/jvi.01436-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 6

Author(s):

Jiangwei Song ◽

Peng Gao ◽

Can Kong ◽

Lei Zhou ◽

Xinna Ge ◽

...

Keyword(s):

Life Cycle ◽

Alveolar Macrophages ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Biological Significance ◽

Hypervariable Region ◽

Supporting Cell ◽

Respiratory Syndrome Virus ◽

Replicase Protein ◽

Cellular Tropism

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global pork production and has been notorious for its rapid genetic evolution in the field. The nonstructural protein 2 (nsp2) replicase protein represents the fastest evolving region of PRRSV, but the underlying biological significance has remained poorly understood. By deletion mutagenesis, we discovered that the nsp2 hypervariable region plays an important role in controlling the balance of genomic mRNA and a subset of subgenomic mRNAs. More significantly, we revealed an unexpected link of the nsp2 hypervariable region to viral tropism. Specifically, a mutant of the Chinese highly pathogenic PRRSV strain JXwn06 carrying a deletion spanning nsp2 amino acids 323 to 521 (nsp2Δ323–521) in its hypervariable region was found to lose infectivity in primary porcine alveolar macrophages (PAMs), although it could replicate relatively efficiently in the supporting cell line MARC-145. Consequently, this mutant failed to establish an infection in piglets. Further dissection of the viral life cycle revealed that the mutant had a defect (or defects) lying in the steps between virus penetration and negative-stranded RNA synthesis. Taken together, our results reveal novel functions of nsp2 in the PRRSV life cycle and provide important insights into the mechanisms of PRRSV RNA synthesis and cellular tropism. IMPORTANCE The PRRSV nsp2 replicase protein undergoes rapid and broad genetic variations in its middle region in the field, but the underlying significance has remained enigmatic. Here, we demonstrate that the nsp2 hypervariable region not only plays an important regulatory role in maintaining the balance of different viral mRNA species but also regulates PRRSV tropism to primary PAMs. Our results reveal novel functions for PRRSV nsp2 and have important implications for understanding the mechanisms of PRRSV RNA synthesis and cellular tropism.

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Functionally active cyclin-dependent kinase 9 is essential for porcine reproductive and respiratory syndrome virus subgenomic RNA synthesis

Molecular Immunology ◽

10.1016/j.molimm.2021.05.004 ◽

2021 ◽

Vol 135 ◽

pp. 351-364

Author(s):

Meng-Di Wang ◽

Le Yang ◽

Jie-Jie Meng ◽

Jia-Jia Pan ◽

Chao Zhang ◽

...

Keyword(s):

Rna Synthesis ◽

Respiratory Syndrome Virus ◽

Cyclin Dependent Kinase ◽

Subgenomic Rna

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Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation

Journal of Virology ◽

10.1128/jvi.80.1.85-94.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 85-94 ◽

Cited By ~ 91

Author(s):

Beau J. Fenner ◽

Rekha Thiagarajan ◽

Hui Kheng Chua ◽

Jimmy Kwang

Keyword(s):

Rna Interference ◽

Reverse Genetics ◽

Nonstructural Protein ◽

Juvenile Fish ◽

Nervous Necrosis Virus ◽

Rna Transfection ◽

B2 Rna ◽

Rna Accumulation ◽

High Level ◽

Late Stages

ABSTRACT Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

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Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3

Journal of Virology ◽

10.1128/jvi.00197-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 6033-6047 ◽

Cited By ~ 12

Author(s):

Kelley R. Hurst-Hess ◽

Lili Kuo ◽

Paul S. Masters

Keyword(s):

Rna Synthesis ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Proteolytic Processing ◽

Alternative Mechanism ◽

Genome Replication ◽

Structural Domains ◽

Subgenomic Rna ◽

Amino Terminal ◽

Viral Nucleocapsid

ABSTRACTCoronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis.IMPORTANCEThe largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.

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A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽

10.3390/v13020289 ◽

2021 ◽

Vol 13 (2) ◽

pp. 289

Author(s):

Kathleen K. M. Glover ◽

Danica M. Sutherland ◽

Terence S. Dermody ◽

Kevin M. Coombs

Keyword(s):

Amino Acid ◽

Temperature Sensitivity ◽

Reverse Genetics ◽

Core Protein ◽

Single Point ◽

Single Amino Acid ◽

Single Point Mutation ◽

Temperature Sensitive ◽

Amino Acid Substitutions ◽

Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

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