mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay

Kroath, Hans; Shatkin, Aaron J.

doi:10.1128/jvi.41.3.1105-1108.1982

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…Also, the A13 fragment binds to wt viral cores at 30 to 33°C with high efficiency, comparable to the efficiency observed in experiments in which intact AIMV RNA 4 is added to the reaction mixture and cleavage is coupled to binding (22). In contrast, the cap 1 structure alone (m7GpppGm) or the cap 1 structure containing only one or two additional nucleotides (e.g., m7GpppGmpUp) binds to detergent-treated wt virions or wt viral cores with low efficiency at 30 to 33°C (at least 100-fold less than the efficiency observed with the A13 fragment) (8,22), although these smaller cap-containing species also bind to the PB2 protein (1, 2). At 30 to 33°C, about seven nucleotides attached to the cap are needed for efficient binding to wt viral cores (22).…”

Section: Discussionmentioning

confidence: 93%

Influenza Virus Temperature-Sensitive Cap (m ⁷ GpppNm)-Dependent Endonuclease

Ulmanen

Broni

Krug

1983

J Virol

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The first step in influenza viral mRNA synthesis is the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m 7 GpppNm) structures to generate capped primers that are 10 to 13 nucleotides long, which are then elongated to form the viral mRNA chains. We examined the temperature sensitivity of these steps in vitro by using two WSN virus temperature-sensitive mutants, ts 1 and ts 6, which have a defect in the genome RNA segment coding for the viral PB2 protein. For these experiments, it was necessary to employ purified viral cores rather than detergent-treated virions to catalyze transcription, as preparations of detergent-treated virions contain destabilizing or inhibitory activities which render even the transcription catalyzed by wild-type virus temperature sensitive. Using purified wild-type viral cores, we found that the rates of endonucleolytic cleavage of capped primers and of overall transcription were similar at 39.5 and 33°C, the in vivo nonpermissive and permissive temperatures, respectively. In contrast, the activities of the cap-dependent endonucleases of ts 1 and ts 6 viral cores at 39.5°C were only about 15% of those at 33°C. The steps in transcription after endonucleolytic cleavage of the capped RNA primer were largely, if not totally, temperature insensitive, indicating that the mutations in the PB2 protein found in ts 1 and ts 6 virions affect only the endonuclease step. The temperature-sensitive defect is most likely in the recognition of the 5′-terminal cap 1 structure that occurs as a required first step in the endonuclease reaction: the cap-dependent binding of a specific capped primer fragment to ts 1 viral cores was temperature sensitive under conditions in which binding to wild-type viral cores was not affected by increasing the temperature from 33 to 39.5°C. Thus, our results establish that the viral PB2 protein functions in cap recognition during the endonuclease reaction.

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