The PA influenza virus polymerase subunit is a phosphorylated protein.

Sanz‐Ezquerro, Juan José; Santarén, Juán F.; Sierra, Teresa Murguı́a de; Aragón, Tomás; Ortega, Joaquı́n; Ortı́n, Juan; Smith, G.L.; Nieto, Amelia

doi:10.1099/0022-1317-79-3-471

Cited by 50 publications

(36 citation statements)

References 40 publications

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“…We have reported previously that the PA polymerase subunit is a phosphorylated protein (38). Moreover, when the phosphorylation state of mutant PAT157A was studied with a vaccinia virus PAT157A recombinant, we observed that this mutant protein was underphosphorylated (32).…”

Section: Rescue Of Recombinant Mutant Virusesmentioning

confidence: 92%

“…However, by mutation analysis, we observed that the ability of PA to induce in vivo protein degradation lay within the 247 N-terminal amino acids of the protein, threonine 157 being involved in this activity (32,39). The PA protein is phosphorylated in vivo and is a substrate of casein kinase II in vitro (38). Mutation of the N-terminal potential casein kinase II phosphorylation sites, especially positions T157 and T162, which are conserved among all strains of influenza type A viruses, led to proteasedeficient PA mutants (32,39).…”

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confidence: 99%

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Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses

Huarte

Falcón

Nakaya

et al. 2003

J Virol

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Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo. In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.The influenza virus RNA polymerase is a complex composed of three subunits, PA, PB1, and PB2. The polymerase, together with the nucleoprotein (NP) and the viral RNA template, form viral ribonucleoproteins (RNPs), which carry out viral transcription and replication. The polymerase complex synthesizes three different RNA classes: (i) mRNAs that contain a cap structure and 10 to 15 nucleotides derived from host cell mRNAs at their 5Ј end and a poly(A) tail at their 3Ј end; (ii) virion RNAs found in the viral particle (vRNAs); and (iii) cRNAs that are full-length complements of vRNA molecules and act as replicative intermediates (19,24).The PB1 subunit contains the polymerase active site, with sequence motifs characteristic of viral RNA-dependent RNA polymerases (34) which are essential for its activity (5). It also contains sites for sequence-specific binding to the conserved 5Ј-and 3Ј-terminal sequences of the vRNA and cRNA molecules (10,11,22). The PB2 protein binds cap1 structures (6, 15, 43). Although previous evidence indicated that the endonucleolytic activity responsible for the cleavage of host mRNA precursors could be contained on this subunit (7, 23), it has been proposed that the endonucleolytic activity lies on the PB1 subunit (21). Because it is responsible for cap-binding activity, PB2 is involved in transcription, but recently, we identified mutations in the PB2 subunit that specifically altered viral RNA replication (9a). The phenotype of viral temperaturesensitive mutants suggests that the PA subunit may be involved in the transition from mRNA transcription to cRNA and vRNA synthesis (reviewed in reference 24), but its precise role in this process is unknown at present. A recent report identified a mutation in the PA protein that shows a phenotype i...

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Section: Rescue Of Recombinant Mutant Virusesmentioning

confidence: 92%

mentioning

confidence: 99%

Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses

Huarte

Falcón

Nakaya

et al. 2003

J Virol

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“…At least four of the different proteins involved in the formation of the ribonucleoprotein complexes (NS1, nucleoprotein, PA, and M1) are phosphoproteins. It has been speculated that the formation of the ribonucleoprotein complexes might be modulated by a phosphorylation-dephosphorylation mechanism (26). We hypothesize that the 23K and 35K forms of the calicivirus ORF3 protein play different roles during virus replication, and identifying these distinct functions is an ongoing endeavor.…”

Section: Discussionmentioning

confidence: 99%

Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions

Glass

Zeng

Estes

2003

J Virol

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Expression of the Norwalk virus open reading frame 3 (ORF3) inSpodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Norwalk virus (NV) is the prototype strain of the Norovirus genus in the family Caliciviridae (24). The noroviruses are a group of viruses that are the major pathogens causing epidemic nonbacterial gastroenteritis (8, 31). The NV genome is a positive-sense, single-stranded RNA molecule approximately 7.7 kb in length predicted to encode three open reading frames (ORFs) (16,18). The first ORF (ORF1) encodes the nonstructural proteins. The second ORF (ORF2) encodes the capsid protein. Expression of the capsid protein in insect cells infected with baculovirus recombinants results in the self assembly of empty recombinant virus-like particles (rVLPs) (17, 32). The third ORF (ORF3) is located at the 3Ј end of the genome and codes for a 212-amino-acid protein that has been characterized as a minor structural protein (9). Expression of the NV ORF3 region in insect cells yields two forms of ORF3 protein, a 23,000-molecular-weight (23K) unphosphorylated form and a 35K phosphorylated form (9). While the ORF3 protein is not essential for the formation of empty VLPs (10,13,17,21,32), the ORF3 23K protein is associated with recombinant NV (rNV) VLPs purified from insect cell cultures infected either with a baculovirus recombinant expressing the entire 3Ј end of the genome or by coinfection with baculovirus recombinants that individually express the ORF2 and ORF3 proteins (9).Protein expression and interaction studies of the human caliciviruses remain limited due to the lack of a tissue culture system for virus propagation. Conservation of ORF3 in all caliciviruses suggests that it performs an important function in the virus life cycle. The ORF3 protein is present in feline calicivirus (FCV) and rabbit hemorrhagic disease virus (RHDV) particles (22,29). However, no studies have examined the potential domains present in the ORF3 protein. In this paper, we report domains of the ORF3 protein responsible for formation of the larger 35K form and for ORF3 proteincapsid protein interaction. In addition, we report further characterization of the 35K form. These studies were facilitated by the generation of baculovirus recombinants that express mutant ORF3 proteins in insect cells and ORF3-specific peptide antisera. The baculovirus recombinants were also coinfected with an ORF2 baculovirus recombinant to examine the ability of the ORF3 mutant proteins to interact with the ORF2 protein allowing incorporation into rNV VLPs. MATERIALS AND METHODSPlasmids and recombinant baculoviruses. For N-terminal HA-tagged ORF3, the hemagglutinin (HA) tag was cloned onto the N terminus of the ORF3 protein by PCR amplification using pri...

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“…The E154G mutation, which arose spontaneously during the cDNA cloning of the PA gene, completely abolished polymerase activity and proteolysis activity (32,33). A mutation at amino acid position 157, which is a potential phosphorylation site, also diminished polymerase activity and proteolysis activity (34)(35)(36). Therefore, we were concerned that the point mutation at position 155 would disrupt some PA functions and in so doing impair virus replication and pathogenicity.…”

Section: Discussionmentioning

confidence: 99%