scholarly journals Hsp90 is required for snakehead vesiculovirus replication via stabilizing the viral L protein

2021 ◽  
Author(s):  
Yanwei Zhang ◽  
Yong-An Zhang ◽  
Jiagang Tu
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Insoluble Fraction ◽  
Viral Rna ◽  
Cellular Proteins ◽  
Economic Losses ◽  
L Protein ◽  
Hsp90 Activity ◽  
The Stability

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. The Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensure the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. Importance It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help understand the polymerase function of negative-stranded RNA viruses.

scholarly journals Oligomerization of Mumps Virus Phosphoprotein

2015 ◽  
Vol 89 (21) ◽  
pp. 11002-11010 ◽  
Author(s):  
Adrian Pickar ◽  
Andrew Elson ◽  
Yang Yang ◽  
Pei Xu ◽  
Ming Luo ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Mutational Analysis ◽  
Mumps Virus ◽  
Viral Rna ◽  
Large Protein ◽  
Terminal Domain ◽  
P Protein ◽  
Minigenome System ◽  
Oligomerization Domain

ABSTRACTThe mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (PN), the oligomerization domain (PO), and the C-terminal domain (PC). While PNis known to relax the NP-bound RNA genome, the roles of POand PCare not clear. In this study, we investigated the roles of POand PCin viral RNA synthesis using mutational analysis and a minigenome system. We found that PNand PCfunctions can betrans-complemented. However, this complementation requires PO, indicating that POis essential for P function. Using thistrans-complementation system, we found that P forms parallel dimers (PNto PNand PCto PC). Furthermore, we found that residues R231, K238, K253, and K260 in POare critical for P's functions. We identified PCto be the domain that interacts with L. These results provide structure-function insights into the role of MuV P.IMPORTANCEMuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented intrans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.

scholarly journals Role of the Conserved Nucleotide Mismatch within 3′- and 5′-Terminal Regions of Bunyamwera Virus in Signaling Transcription

2005 ◽  
Vol 79 (6) ◽  
pp. 3586-3594 ◽  
Author(s):  
John N. Barr ◽  
Gail W. Wertz
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Nucleotide Identity ◽  
Nucleotide Position ◽  
Transcription Activity ◽  
Nucleotide Mismatch ◽  
Negative Sense ◽  
Bunyamwera Virus ◽  
Transcription And Replication

ABSTRACT Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of tri-partite negative-sense RNA viruses. The three BUNV segments possess 3′ and 5′ nontranslated regions (NTRs) that signal two RNA synthesis activities: (i) transcription to generate mRNAs and (ii) replication to generate antigenomes that are replicated to yield further genomes. While the genome acts as a template for synthesis of both transcription and replication products, the antigenome allows synthesis of only replication products, with mRNAs being undetectable. Here, we investigate the basis for the fundamentally different signaling abilities of genomic and antigenomic strands. We show that the identity of only nucleotide position 9 within the genomic 3′ NTR is critical for the different RNA synthesis characteristics of genomic and antigenomic strands, thus identifying this nucleotide as an essential component of the transcription promoter. This nucleotide is distinctive, as it interrupts an unbroken run of conserved complementary nucleotides within the 3′ and 5′ NTRs of all three segments. Our results show that the conserved mismatched arrangement of this nucleotide plays no detectable role in signaling transcription. Instead, we show that the transcription-signaling ability of this position is entirely dependent on its nucleotide identity. We further show that while a U residue at 3′ position 9 is strongly preferred for transcription activity in the context of the genomic promoter, it does not signal transcription in the context of the antigenomic promoter. Therefore, our results show that the identity of 3′ position 9 is crucial for signaling BUNV transcription; however, it is not the sole determinant.

scholarly journals Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Philip V'kovski ◽  
Markus Gerber ◽  
Jenna Kelly ◽  
Stephanie Pfaender ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Viral Rna ◽  
Host Proteins ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Starting Point ◽  
Positive Strand Rna

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

Role of GS-5734 (Remdesivir) in inhibiting SARS-CoV and MERS-CoV: The expected role of GS-5734 (Remdesivir) in COVID-19 (2019-nCoV) - VYTR hypothesis.

2020 ◽  
Vol 11 (SPL1) ◽  
Author(s):  
Vityala Yethindra
Keyword(s):  
Clinical Trials ◽  
Antiviral Activity ◽  
Protease Inhibitors ◽  
Human Body ◽  
Broad Spectrum ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Improved Outcomes ◽  
The Family

Coronaviruses (CoVs) are enveloped RNA viruses related to the family Coronaviridae, the order Nirdovales, and observed in humans and other mammals. In December 2019, many pneumonia cases reported by patients with unknown causes, mainly associated with seafood and wet animal market in Wuhan, China, and where clinically resembled viral pneumonia. At present, there is no existence of antiviral drugs for the treatment of CoV infections. The results of our study are GS-5734 strongly inhibits SARS-CoV and MERS-CoV in HAE cells, GS-5734 inhibits CoVs at early stages in replication by inhibiting viral RNA synthesis, the absence of ExoN-mediated proofreading in viruses sensitive to treatment with GS-5734. Protease inhibitors can show improved outcomes in some coronaviruses, but mostly 99% of protease inhibitors bind to proteins present in the human body, and only 1% attacks on existed viruses. The expected role of GS-5734 (Remdesivir) in the 2019-nCoV - VYTR hypothesis explained. As broad-spectrum drugs are capable of inhibiting CoV infections, GS-5734 is a broad-spectrum drug and may show inhibition on CoV infections and 2019-nCoV. GS-5734 will show desired results regarding antiviral activity against 2019-nCoV as it showed potent antiviral activity in other CoVs. More clinical trials and experiments needed to prove that GS-5734 (Remdesivir) is a potential and effective drug to treat 2019-nCoV.

scholarly journals Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (9) ◽  
pp. 5136-5144 ◽  
Author(s):  
B. Joan Morasco ◽  
Nidhi Sharma ◽  
Jessica Parilla ◽  
James B. Flanegan
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Positive Strand ◽  
Negative Strand ◽  
Replication Model ◽  
Covalently Linked

ABSTRACT The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive- but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3′ poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.

scholarly journals Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Ryan H. Gumpper ◽  
Weike Li ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Codon Usage ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Genomic Rna ◽  
Negative Strand ◽  
Unique Mechanism

ABSTRACTNegative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. Byin situanalyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis.IMPORTANCENegative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

scholarly journals Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Helena J. Maier ◽  
Philippa C. Hawes ◽  
Eleanor M. Cottam ◽  
Judith Mantell ◽  
Paul Verkade ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Infectious Bronchitis ◽  
Positive Sense ◽  
Infected Cells

ABSTRACTReplication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells ofex vivotracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.IMPORTANCEAll positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

scholarly journals Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

2016 ◽  
Vol 90 (7) ◽  
pp. 3676-3683 ◽  
Author(s):  
James R. Short ◽  
Jeffrey A. Speir ◽  
Radhika Gopal ◽  
Logan M. Pankratz ◽  
Jason Lanman ◽  
...  
Keyword(s):  
Host Defense ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Flock House Virus ◽  
Double Stranded Rna ◽  
Content Type ◽  
Positive Sense

ABSTRACTViruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infectsDrosophilacells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infectedDrosophilacells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infectedDrosophilacells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

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 Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2540
Author(s):  
Nicole Doyle ◽  
Jennifer Simpson ◽  
Philippa C. Hawes ◽  
Helena J. Maier
Keyword(s):  
Life Cycle ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Viral Rna ◽  
Host Cells ◽  
Infectious Bronchitis ◽  
Welfare Issue ◽  
Membrane Bound ◽  
Positive Strand Rna

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

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