Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression

Abstract

To determine the function(s) of the PB2 protein of influenza A virus, six temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each carrying a ts mutation in the PB2 gene, were analysed for virus RNA and protein synthesis. One of the mutants, ICRC27, exhibited unique phenotypes and was characterized in detail. At the non-permissive temperature, 40 °C, the accumulation of mRNA for each genome segment was reduced severely, leading to delayed and reduced synthesis of viral proteins, complementary and viral RNAs (cRNAs and vRNAs). At the permissive temperature, 34 °C, the mutant virus produced severalfold greater concentrations of both mRNAs and cRNAs of PB2, PB1 and PA segments than wild-type virus. The synthesis of the three polymerase proteins and the induction of RNA polymerase activity were also greatly increased. By contrast, the expression of the haemagglutinin (HA) gene was severely suppressed. The over-production of the polymerase mRNAs was not observed during primary transcription, i.e. in the presence of cycloheximide. The ts ÷ revertants oflCRC27 did not exhibit the ts defects and also lost most of the non-ts phenotypes at 34 °C. These observations indicate that the PB2 protein participates not only in the synthesis of viral RNAs, but also in the regulation of viral gene expression, i.e. in the downregulation of the three polymerase genes and the upregulation of the HA gene during secondary transcription.

Mutational Analysis of the Uukuniemi Virus ( Bunyaviridae Family) Promoter Reveals Two Elements of Functional Importance

“…The vRNA/cRNA ratio of about 9:1 obtained for the wild-type promoter construct was expected, since synthesis of cRNA, a replicative intermediate, in most viral systems is substantially lower than that of vRNA synthesis. This result was similar to that obtained for influenza A virus, where a vRNA/ cRNA ratio of 10:1 was demonstrated (30). However, only a minor influence on the vRNA/cRNA ratio could be detected for the mutants tested.…”

“…As expected, the vRNA amplification product was easily detectable for all examined promoter mutants due to the RNA transcribed by RNA polymerase I. However, the amount of cRNA was much lower than that of the vRNA, as expected for a negative-strand RNA virus (30) (Fig. 5, upper panel).…”

Observations of Immuno-Gold Conjugates on Influenza Viruses Using Waveguide-Mode Sensors

“…In brief, viral proteins were separated by SDS-polyacrylamide gel electrophoresis as described before [41] and transferred to polyvinylidene difluoride membrane. After immobilization of the protein on the membrane, the membrane was blocked with 3% nonfat milk in TBS buffer at room temperature for 1 h and then treated with a 1∶10,000 dilution of the primary antibody (anti-Udorn antibody) at 4°C overnight.…”

Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit

“…It has been suggested that the PB2 subunit acts as an endonuclease in the generation of transcription primers (26), but the enzymatic activity requires the association of all three polymerase subunits and both template RNA ends (16), supporting the notion that the active viral ribonucleoproteins are in a panhandle structure (17). A number of reports have described the isolation and characterization of TS PB2 gene mutants (23,25,29,30,34,50,54). These mutants showed a block in mRNA synthesis and hence in virion RNA and protein accumulation, and one of the mutants described (34) showed an altered pattern of mRNA synthesis at the permissive temperature.…”