A temperature-sensitive mutation in the acidic polymerase gene of an influenza A virus alters the regulation of viral protein synthesis

Herget, Martina; Scholtissek, Christoph

doi:10.1099/0022-1317-74-9-1789

Cited by 6 publications

(1 citation statement)

References 34 publications

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“…7). Even though fine mapping has not yet been performed, the ts263 mutation of PA, which affects genome replication, has been mapped to this region (amino acid position 671; Herget & Scholtissek, 1993).…”

Section: Subunit-subunit Contact Sites On Each P Proteinmentioning

confidence: 99%

Molecular Assembly of the Influenza Virus RNA Polymerase: Determination of the Subunit-Subunit Contact Sites

Toyoda

Adyshev

Iwata

et al. 1996

Journal of General Virology

104

114

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Journal ' oj~ G~nera(. V!ro!og.y.. (.! .9.96!., 7.1~..2 .! .49-2157 .. P!!n!.e(J. i.rl..C~r~eat. B r!!a! n ......................................................................................................................... Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunitsubunit contact sites on all three of the P proteins were determined. Results indicate that binary complexes are formed between PB1-PB2 and PB1-PA but not between PB2-PA. Therefore, we concluded that PB 1 is the core subunit for assembly of the virus RNA polymerase. The C-terminal 1 58 amino acids of PB1 bound to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two-thirds of PA. PB2-PA binding was not detected when they were expressed in the absence of the PB1 subunit. Molecular assembly of the influenza virus

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Section: Subunit-subunit Contact Sites On Each P Proteinmentioning

confidence: 99%

Molecular Assembly of the Influenza Virus RNA Polymerase: Determination of the Subunit-Subunit Contact Sites

Toyoda

Adyshev

Iwata

et al. 1996

Journal of General Virology

104

114

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

et al. 1999

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Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome, including the unique cap-I-dependent RNase activity. To map the important domains for RNA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we generated site-specific antibodies against overlapping 150-amino-acid peptides that cover each entire subunit. Monospecific antibodies against each subunit inhibited RNA synthesis in vitro. Those against PB1 and PB2 inhibited the cap-I-dependent RNase activity, but those against PB2 alone slightly inhibited the cap-I-binding activity. Antibodies against the N-terminal amino acids 1-159 of PB2 that overlap the PB1-binding site on PB2 and the C-terminal amino acids 501-617 of PA that overlap the putative nucleotide-binding site and PB1-binding site on PA inhibited RNA polymerizing activity as well as monospecific antibodies. Those against the N-terminal (amino acids 1-159); the central region (amino acids 305-559) of PB2, where a part of the cap-binding domain predicted previously is localized; the N-terminal (amino acids 1-222) of PB1; and amino acids 301-517 and 601-716 of PA inhibited the cap-I-dependent RNase activity. The cap-binding domain on PB2 could be mapped in amino acids 402-559, where one of the cap-binding domains mapped previously overlapped.

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The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

Chan¹,

Zhou²,

Kemble³

et al. 2008

Virology

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We have previously determined that the temperature sensitive (ts) and attenuated (att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 degrees C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 degrees C during a single-step replication, however, vRNA synthesis was reduced and the nuclear-cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 degrees C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle.

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A temperature-sensitive mutation in the acidic polymerase gene of an influenza A virus alters the regulation of viral protein synthesis

Abstract: The temperature-sensitive defect of mutant ts 263

Cited by 6 publications

References 34 publications

Molecular Assembly of the Influenza Virus RNA Polymerase: Determination of the Subunit-Subunit Contact Sites

Molecular Assembly of the Influenza Virus RNA Polymerase: Determination of the Subunit-Subunit Contact Sites

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

The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

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