scholarly journals Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription

2003 ◽  
Vol 77 (9) ◽  
pp. 5098-5108 ◽  
Author(s):  
Pablo Gastaminza ◽  
Beatriz Perales ◽  
Ana M. Falcón ◽  
Juan Ortín
Keyword(s):  
Influenza Virus ◽  
Rna Replication ◽  
Terminal Region ◽  
Viral Rna ◽  
Mrna Accumulation ◽  
Virus Rna ◽  
Transcription In Vitro ◽  
Type Mutant

ABSTRACT PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

scholarly journals Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2337-2348 ◽  
Author(s):  
Tao Deng ◽  
Frank T. Vreede ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
De Novo ◽  
Viral Rna ◽  
Virus Rna ◽  
Initiation Of Replication ◽  
Replicative Intermediates ◽  
Internal Initiation

ABSTRACT Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5′ triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

scholarly journals Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

2005 ◽  
Vol 79 (11) ◽  
pp. 6631-6643 ◽  
Author(s):  
Diego E. Alvarez ◽  
María F. Lodeiro ◽  
Silvio J. Ludueña ◽  
Lía I. Pietrasanta ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Virus Genome ◽  
Viral Rna ◽  
Physical Interaction ◽  
Virus Rna ◽  
Rna Elements ◽  
Rna Interaction

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

scholarly journals Mechanism of Action of T-705 against Influenza Virus

2005 ◽  
Vol 49 (3) ◽  
pp. 981-986 ◽  
Author(s):  
Yousuke Furuta ◽  
Kazumi Takahashi ◽  
Masako Kuno-Maekawa ◽  
Hidehiro Sangawa ◽  
Sayuri Uehara ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Mdck Cells ◽  
Specific Inhibitor ◽  
Polymerase Activity ◽  
Virus Activity ◽  
Virus Rna ◽  
Cellular Dna

ABSTRACT T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5′-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5′-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.

scholarly journals Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1α is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication

2007 ◽  
Vol 88 (10) ◽  
pp. 2800-2810 ◽  
Author(s):  
Michela Campagna ◽  
Mauricio Budini ◽  
Francesca Arnoldi ◽  
Ulrich Desselberger ◽  
Jorge E. Allende ◽  
...  
Keyword(s):  
Cytoplasmic Inclusion ◽  
Rna Replication ◽  
Viral Rna ◽  
Structural Protein ◽  
Cytoplasmic Inclusion Bodies ◽  
Replicative Cycle ◽  
In Cells

The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1α is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1α is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1α was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1α is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection.

scholarly journals Viability of Poliovirus/Rhinovirus VPg Chimeric Viruses and Identification of an Amino Acid Residue in the VPg Gene Critical for Viral RNA Replication

2003 ◽  
Vol 77 (13) ◽  
pp. 7434-7443 ◽  
Author(s):  
I. Wayne Cheney ◽  
Suhaila Naim ◽  
Jae Hoon Shim ◽  
Meghan Reinhardt ◽  
Bharati Pai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Rna Replication ◽  
Viral Rna ◽  
Sequencing Analysis ◽  
Virus Particles ◽  
Chimeric Rna ◽  
Chimeric Viruses

ABSTRACT Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3Dpol, in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3Dpol-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3Dpol was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3Dpol could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3Dpol to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu6 to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3Dpol in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.

scholarly journals CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Yu-Chen Lin ◽  
King-Song Jeng ◽  
Michael M. C. Lai
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Ubiquitin Ligase ◽  
Influenza A ◽  
Rna Binding ◽  
Rna Replication ◽  
Viral Rna ◽  
Mass Spectrometry Analysis ◽  
Positive Role

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro -translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. IMPORTANCE Influenza virus, particularly influenza A virus, causes severe and frequent outbreaks among human and avian species. Finding potential target sites for antiviral agents is of utmost importance from the public health point of view. We previously found that viral nucleoprotein (NP) is ubiquitinated, and ubiquitination enhances viral RNA replication. In this study, we found a cellular ubiquitin ligase, CNOT4, capable of ubiquitinating NP. The ubiquitination sites are scattered on the surface of the NP molecule, which is critical for RNA replication. CNOT4 and a ubiquitin protease, USP11, together regulate the extent of NP ubiquitination and thereby the efficiency of RNA replication. This study thus identifies a potential antiviral target site and reveals a novel posttranslational mechanism for regulating viral replication. This represents a novel finding in the literature of influenza virus research.

scholarly journals Tyrosine 3 of Poliovirus Terminal Peptide VPg(3B) Has an Essential Function in RNA Replication in the Context of Its Precursor Protein, 3AB

2007 ◽  
Vol 81 (11) ◽  
pp. 5669-5684 ◽  
Author(s):  
Ying Liu ◽  
David Franco ◽  
Aniko V. Paul ◽  
Eckard Wimmer
Keyword(s):  
Rna Replication ◽  
Precursor Protein ◽  
Viral Rna ◽  
Hydroxyl Group ◽  
Wild Type ◽  
Replication Complex ◽  
A Genome ◽  
Essential Function

ABSTRACT Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3Dpol, but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3Dpol. Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.

scholarly journals Influenza A Virus RNA Polymerase Has the Ability To Stutter at the Polyadenylation Site of a Viral RNA Template during RNA Replication

1999 ◽  
Vol 73 (6) ◽  
pp. 5240-5243 ◽  
Author(s):  
Hongyong Zheng ◽  
Hye Annie Lee ◽  
Peter Palese ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Viral Polymerase ◽  
Virus Rna ◽  
Independent Synthesis ◽  
New Evidence ◽  
Virus Mrnas

ABSTRACT The viral polymerase of influenza virus, a negative-strand RNA virus, is believed to polyadenylate the mRNAs by stuttering at a stretch of five to seven uridine residues which are located close to the 5′ ends of the viral RNA templates. However, a mechanism of polyadenylation based on a template-independent synthesis of the poly(A) tail has not been excluded. In this report, we present new evidence showing the inherent ability of the viral polymerase to stutter at the poly(U) stretch of a viral RNA template during RNA replication. Variants which possess 1- to 13-nucleotide-long insertions at the poly(U) stretch have been identified. These results support a stuttering mechanism for the polyadenylation of influenza virus mRNAs.

scholarly journals Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2162-2169 ◽  
Author(s):  
Saulius Serva ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Rna Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Mass Spectrometry Analysis ◽  
Host Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Viral Replicase

ABSTRACT Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown ∼35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.

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