PA Subunit from Influenza Virus Polymerase Complex Interacts with a Cellular Protein with Homology to a Family of Transcriptional Activators

Huarte, Maite; Sanz‐Ezquerro, Juan José; Roncal, Fernando; Ortı́n, Juan; Nieto, Amelia

doi:10.1128/jvi.75.18.8597-8604.2001

Cited by 114 publications

(141 citation statements)

References 54 publications

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“…In this regard, the proteolytic activity of the PA subunit and/or its phosphorylation state might be important to allow potential changes in the conformation of the polymerase. Finally, interaction of the PA subunit with hCLE, a human protein homologous to transcription activation factors, has been reported in vitro and in vivo in reconstituted RNPs (16). Although PAT157A interacts with hCLE with the same strength as PAwt, as measured by two-hybrid analysis (16), we cannot exclude that mutations at positions T157 alter the interaction of the polymerase with hCLE or other cellular factors required for viral RNA replication.…”

Section: Discussionmentioning

confidence: 98%

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: Discussionmentioning

confidence: 98%

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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“…Likewise, binding to the cRNA promoter may imply structural alterations, because vRNA and cRNA panhandles are recognized in different ways by the polymerase (22). On the other hand, the replicating polymerase could present a number of additional structural alterations as a consequence of its interaction with soluble NP or with cellular factors like hCLE (49) or Hsp90 (50).…”

Section: Mapping Pb1 Subunit Domains By 3d Reconstruction Of Taggedmentioning

confidence: 99%

3D structure of the influenza virus polymerase complex: Localization of subunit domains

Area‐Gómez

Martín‐Benito

Gastaminza

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

114

112

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The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.M any viruses contain RNA as genetic material and use RNA-dependent RNA polymerases as enzymes for replication and transcription. The atomic structures of several viral RNA (vRNA)-dependent RNA polymerases from positive-and double-stranded RNA viruses are available (1-6). These proteins show little sequence homology with other DNA-dependent polymerases but contain specific sequence motifs shared with other polymerases (7). They show a typical right-hand fold that includes the thumb, finger, and palm domains, the last of which contains the conserved sequence motifs involved in polymerase catalysis. These polymerases are medium-size proteins that show activity in vitro, although viral replication and transcription in vivo are carried out by polymerase-containing macrocomplexes. In these, other viral and cellular factors contribute to the efficiency and regulation of the polymerase or to the localization of the RNA synthetic machinery in the infected cell (reviewed in ref. 8).The situation in negative-strand viruses (NSVs) is different. The templates for transcription and replication are ribonucleoproteins (RNPs) in which the negative-stranded RNA is complexed with the nucleoprotein (NP) and associated to the polymerase. In the NSVs, the polymerase is a very large protein, the L protein (Ϸ 250 kDa, except in the Orthomyxoviridae), which consists of a heterotrimer of similar aggregate size. The replication intermediates are RNPs similar to virion RNPs but containing positive-stranded RNA. Other viral proteins, such as the P protein for nonsegmented NSVs, and cellular factors are essential for transcription or replication of NSV RNPs. As a consequence of this complexity, very little structural information is available on NSV polymerases.Within the NSVs, influenza A viruses are distinct in having a segmented genome and replicating in the cell nucleus (9). Their genome consists of eight RNPs that are transcribed and replicated by the viral polymerase, a heterotrimer composed by the PB1, PB2, and PA subunits (10, 11). Replication of vRNA involves de novo initiation of nascent RNA chains, whereas initiation of mRNA synthesis requires the generation of capped primers from cellular heterogeneous nuclear RNAs by a capstealing mechanism (12). Transcript...

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“…13 The majority of the above described cellular factors were identified by either yeast-two-hybrid screens with individual components of vRNP, including polymerase subunits, or coimmunoprecipitation experiments using antibodies directed against a suspected cellular interaction partner. 5,8,9,12,13 It is therefore reasonable to assume that not all interaction partners have been identified, especially those proteins interacting with native vRNPs or polymerase complexes. To identify cellular factors associated to these native viral complexes we purified reconstituted vRNPs by Strep-tagged viral nucleoprotein (NP-Strep) and the viral polymerase by tandem-affinity-purification (TAP)-tagged polymerase subunits from human cells and analyzed the co-purified cellular factors by mass spectrometry (MS).…”

Section: Introductionmentioning

confidence: 99%

Identification of Cellular Interaction Partners of the Influenza Virus Ribonucleoprotein Complex and Polymerase Complex Using Proteomic-Based Approaches

et al. 2006

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SummaryCellular factors which associate with the influenza A viral ribonucleoprotein (vRNP) are presumed to play important roles in the viral life cycle. To date, interaction screens using individual vRNP components, such as the nucleoprotein or viral polymerase subunits, have revealed few cellular interaction partners. To improve this situation, we performed comprehensive, proteomics-based screens to identify cellular factors associated with the native vRNP and viral polymerase complexes. Reconstituted vRNPs were purified from human cells using Strep-tagged viral nucleoprotein (NPStrep) as bait, and co-purified cellular factors were identified by mass spectrometry (MS). In parallel, reconstituted native influenza A polymerase complexes were isolated using tandem affinity purification (TAP)-tagged polymerase subunits as bait, and co-purified cellular factors were again identified by MS. Using these techniques, we identified 41 proteins which co-purified with NP-Strepenriched vRNPs, and 4 cellular proteins which co-purified with the viral polymerase complex. Two of the polymerase-associated factors, importin-β3 and PARP-1, represent novel interaction partners. Most cellular proteins previously shown to interact with either viral NP and/or vRNP were also identified using our method, demonstrating its sensitivity. Co-immunoprecipitation studies in virusinfected cells using selected novel interaction partners, including nucleophosmin (NPM), confirmed their association with vRNP. Immunofluorescence analysis further revealed that NPM is recruited to sites of viral transcription and replication in infected cells. Additionally, overexpression of NPM resulted in increased viral polymerase activity, indicating its role in viral RNA synthesis. In summary, the proteomics-based approaches used in this study represent powerful tools to identify novel vRNPassociated cellular factors for further characterization.

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PA Subunit from Influenza Virus Polymerase Complex Interacts with a Cellular Protein with Homology to a Family of Transcriptional Activators

Cited by 114 publications

References 54 publications

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

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

3D structure of the influenza virus polymerase complex: Localization of subunit domains

Identification of Cellular Interaction Partners of the Influenza Virus Ribonucleoprotein Complex and Polymerase Complex Using Proteomic-Based Approaches

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