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

Abstract

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.

Seasonality and selective trends in viral acute respiratory tract infections

“…This is similar to the optimum temperature of the polymerase of influenza C, which is 33°C [62,63]. Ulmanen et al [64] found that the rate of transcription by detergent-treated WSN viruses (influenza A) was about 10 times greater at 33°C than at 39.5°C, and also that the binding of a cleaved primer cap (the ''A13 fragment") to the viral cores was ''unexpectedly" much weaker at 39.5°C than at 33°C. Scholtissek and Rott [65] showed that the optimum for the polymerase of the Rostock strain of fowl plague virus was 36°C, five degrees below chickens' normal body temperature (41°C).…”

“…There is extensive biochemical evidence for the temperature sensitivity in respiratory viruses, particularly influenza, which is discussed below. This includes temperature sensitivity that is associated with the steps of the uptake of virions into cells [60], RNA transcription [61][62][63][64][65] (including the ts control of the balance between the synthesis of mRNA and replicative products [66,67]), and the transport to the cell surface of hemagglutininesterase-fusion protein, the protein that promotes the fusion of influenza C virions with cells [68].…”

“…6). Moreover, biochemical studies show that many steps in the replication of laboratory strains of influenza and other viruses have residual natural temperature sensitivity [60][61][62][63][64][65][66][67][68]83], in spite of many cycles of replication of most laboratory viruses at 37°C. We can explain these data by suggesting a link between loss of temperature sensitivity and the acquisition of increased virulence in respiratory viruses, which seems to be a legacy of their evolutionary history.…”

Antagonistic coevolution limits population persistence of a virus in a thermally deteriorating environment

“…We chose to manipulate temperature because high temperature often constitutes a significant stress for viruses (probably due to the lower thermostability of their simple viral proteins) but not their hosts (e.g. Ulmanen et al 1983). We created a gradually, rather than abruptly, degrading thermal environment to maximize biological reality (e.g.…”

Chapter 3 Antiviral drugs: general considerations

“…Interestingly, activation by cleavage of a dinucleotide containing one toxic nucleoside has been suggested as a way to utilize viral nucleases as targets for antiviral drugs . Inhibition of the cap dependent endonuclease activity of influenza virus (Ulmanen et al, 1983) should constitute a logical point of attack for an antiviral drug and prevent the utilization of the cellular mRNA cap by the viral polymerase.…”