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

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

scholarly journals 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

2021 ◽  
Author(s):  
Yunhao Hu ◽  
Purui Ke ◽  
Peng Gao ◽  
Yongning Zhang ◽  
Lei Zhou ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Interaction Network ◽  
Single Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Subgenomic Rna ◽  
Linker Region ◽  
Transcription Complexes ◽  
Rna Accumulation

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.

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

2006 ◽  
Vol 80 (6) ◽  
pp. 3108-3111 ◽  
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.

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

2016 ◽  
Vol 90 (11) ◽  
pp. 5384-5398 ◽  
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.

scholarly journals The 5′UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins

Virology ◽  
2009 ◽  
Vol 387 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Raghavendran Kulasegaran-Shylini ◽  
Varatharasa Thiviyanathan ◽  
David G. Gorenstein ◽  
Ilya Frolov
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Subgenomic Rna ◽  
Specific Mutation ◽  
Encoded Proteins

scholarly journals Identification of porcine reproductive and respiratory syndrome virus ORF1a-encoded non-structural proteins in virus-infected cells

2012 ◽  
Vol 93 (4) ◽  
pp. 829-839 ◽  
Author(s):  
Yanhua Li ◽  
Ali Tas ◽  
Eric J. Snijder ◽  
Ying Fang
Keyword(s):  
Rna Synthesis ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antibodies ◽  
Structural Proteins ◽  
Respiratory Syndrome Virus ◽  
Replicase Gene ◽  
Future Studies ◽  
Swine Pathogen ◽  
Infected Cells

The porcine reproductive and respiratory syndrome virus (PRRSV) replicase gene consists of two large ORFs, ORF1a and ORF1b, the latter of which is expressed by ribosomal frameshifting. The ORF1a-encoded part of the resulting replicase polyproteins (pp1a and pp1ab) is predicted to be processed proteolytically into ten non-structural proteins (nsps), known as nsp1–8, with both the nsp1 and nsp7 regions being cleaved internally (yielding nsp1α and nsp1β, and nsp7α and nsp7β, respectively). The experimental verification of these predictions depends strongly on the ability to identify individual cleavage products with specific antibodies. In this study, a panel of monoclonal and polyclonal antibodies was generated, which together were able to recognize eight ORF1a-encoded PRRSV nsps. Using these reagents, replicase cleavage products were detected in PRRSV-infected MARC-145 cells using a variety of immunoassays. By immunofluorescence microscopy, most nsps could be detected by 6 h post-infection. During the early stages of infection, nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8 co-localized in distinct punctate foci in the perinuclear region of the cell, which were determined to be the site of viral RNA synthesis by in situ labelling. Western blot and immunoprecipitation analysis identified most individual nsps and several long-lived processing intermediates (nsp3–4, nsp5–7, nsp5–8 and nsp3–8). The identification and subcellular localization of PRRSV nsps in virus-infected cells documented here provides a basis for the further structure–function studies. Thus, this PRRSV antibody panel will be an important tool for future studies on the replication and pathogenesis of this major swine pathogen.

scholarly journals nsP4 is a major determinant of alphavirus replicase activity and template selectivity

2021 ◽  
Author(s):  
Laura Sandra Lello ◽  
Koen Bartholomeeusen ◽  
Sainan Wang ◽  
Sandra Coppens ◽  
Rennos Fragkoudis ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Subgenomic Rna ◽  
Positive Strand ◽  
Novel Approach ◽  
Positive Strand Rna ◽  
Chimeric Viruses

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the non-structural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1-nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis -active sequences. Here we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123 while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. Importance. A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.

scholarly journals New insights into RNA packaging in porcine reproductive and respiratory syndrome virus

2011 ◽  
Vol 92 (12) ◽  
pp. 2865-2870 ◽  
Author(s):  
Tayyba T. Baig ◽  
Alexander Zakhartchouk
Keyword(s):  
Synthetic Peptide ◽  
Basic Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Binding Region ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Subgenomic Rna ◽  
N Protein ◽  
Virus Particles ◽  
Rich Domain

While identifying whether the smallest packaged heteroclite subgenomic RNA (S9) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a packaging signal, we found that S9 was capable of binding to the basic amino acid-rich domain (synthetic peptide of aa 34–53) of the packaging protein (N). In addition, by using truncations at the 5′ and 3′ ends of S9, a minimal binding region of 35 nt was found to be essential for binding to both the synthetic peptide and to the full-length N protein. Furthermore, by using cell-culture experiments, we found that S9 was capable of packaging non-viral RNA sequence into PRRSV particles and that the 35 nt region was essential for this activity. Taken together, our data suggest that this 35 nt region might be an important element for packaging PRRSV genomic RNA into virus particles.

scholarly journals Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

2007 ◽  
Vol 81 (12) ◽  
pp. 6356-6368 ◽  
Author(s):  
Eric F. Donaldson ◽  
Amy C. Sims ◽  
Rachel L. Graham ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Infectious Clone ◽  
Proteolytic Processing ◽  
Central Core ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Murine Hepatitis Virus ◽  
Mutant Phenotypes

ABSTRACT Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually ∼15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.

scholarly journals Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

2004 ◽  
Vol 78 (12) ◽  
pp. 6091-6101 ◽  
Author(s):  
K. Sivakumaran ◽  
Seung-Kook Choi ◽  
Masarapu Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Core ◽  
Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

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