Molecular Mapping of Influenza Virus RNA Polymerase by Site-Specific Antibodies

Masunaga, Kenji; Mizumoto, Kiyohisa; Kato, Hirohisa; Ishihama, Akira; Toyoda, Tetsuya

doi:10.1006/viro.1999.9625

Cited by 17 publications

(12 citation statements)

References 49 publications

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“…1 and 5). The in vitro interaction data indicate that the hCLE interaction domain maps in regions of PA protein included in its C-terminal third, close to the region of PA that interacts with the PB1 subunit of the polymerase complex (14,27). Correspondingly, a PA mutant with a deletion of 154 amino acids at its N terminus possesses hCLE binding capacity in the two-hybrid system.…”

Section: Discussionmentioning

confidence: 92%

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

Huarte

Sanz‐Ezquerro

Roncal

et al. 2001

J Virol

114

136

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The PA subunit of the influenza virus polymerase complex is a phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from Gallus gallus called CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.The genome of influenza A virus consists of a set of eight single-stranded RNA segments of negative polarity. These RNAs form ribonucleoproteins (RNPs) with four viral proteins: the nucleoprotein (NP) and the three subunits of the polymerase (PB1, PB2, and PA). These elements are required for both transcription and replication of the viral genome (10,16,18,29).The roles of the polymerase subunits have been partly outlined. The PB1 subunit contains sequence motifs typical of the viral RNA-dependent RNA polymerases (43), which have been shown to be essential for RNA synthesis (3), suggesting that this subunit is the polymerase itself. PB2 protein binds to CAP1 structures (4, 51) and is involved in the endonucleolytic cleavage of cellular mRNAs to generate the precursors used as primers for the viral transcription (6,22). PA is a phosphoprotein in vivo and is a substrate of casein kinase II in vitro (47). This subunit induces a proteolytic process when expressed individually, affecting both coexpressed proteins and PA protein itself (46). The amino-terminal third of the molecule is sufficient to activate this proteolysis (48). Recently, we have reconstituted RNPs in vivo from cloned genes using PA point mutants deficient in proteolytic activity. These mutant RNPs are as active as the wild type in their transcription activity but have a lower capacity to support replication of model vRNA ...

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Section: Discussionmentioning

confidence: 92%

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

Huarte

Sanz‐Ezquerro

Roncal

et al. 2001

J Virol

114

136

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“…Therefore, PB1 13P and 678N could improve the interplay of polymerase subunits and NP in a new host environment. SC35M PB2 333I is located within the cap-binding region (30) and might therefore affect viral transcription. The virus without this mutation, SC35M-PB2 333T , exceeds even the high virulence of SC35M.…”

Section: Discussionmentioning

confidence: 99%

The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host

Gabriel

Dauber

Wolff

et al. 2005

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

617

648

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Mammalian influenza viruses are descendants of avian strains that crossed the species barrier and underwent further adaptation. Since 1997 in southeast Asia, H5N1 highly pathogenic avian influenza viruses have been causing severe, even fatal disease in humans. Although no lineages of this subtype have been established until now, such repeated events may initiate a new pandemic. As a model of species transmission, we used the highly pathogenic avian influenza virus SC35 (H7N7), which is low-pathogenic for mice, and its lethal mouse-adapted descendant SC35M. Specific mutations in SC35M polymerase considerably increase its activity in mammalian cells, correlating with high virulence in mice. Some of these mutations are prevalent in chicken and mammalian isolates, especially in the highly pathogenic H5N1 viruses from southeast Asia. These activity-enhancing mutations of the viral polymerase complex demonstrate convergent evolution in nature and, therefore, may be a prerequisite for adaptation to a new host paving the way for new pandemic viruses.evolution ͉ pathogenicity

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“…4). Furthermore, a peptide-speci®c polyclonal antibody againt a PB2 fragment (residues 402±559) inhibits capped RNA-dependent RNA synthesis and capped RNA-crosslinking to PB2 (Masunaga et al 1999). Some in¯uenza virus mutants harbouring mutations in this region renders viruses less pathogenic (Leu512Ile mutation) (McCauley & Penn 1990) and temperature-sensitive growth (Asp556Asn) (Lawson et al 1992) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

1999

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Background: In¯uenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymeraseassociated capped RNA endonuclease. To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32 P only at the cap-1 structure.

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Molecular Mapping of Influenza Virus RNA Polymerase by Site-Specific Antibodies

Cited by 17 publications

References 49 publications

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

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

The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

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