Threonine 157 of influenza virus PA polymerase subunit modulates RNA replication in infectious viruses

Abstract

Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo. In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.

FIG. 1.

Multiplication of recombinant viruses. MDCK cells were infected at either high (A) or low (B) multiplicity. At the indicated times, aliquots were taken, and virus infectivity was evaluated by plaque assay.

FIG. 2.

Kinetics of nuclear transport of wild-type and mutant PA proteins in infected cells. Cultures of MDCK cells were infected with wild-type and PA mutant viruses. At the indicated times, the cultures were fixed and processed for immunofluorescence with PA- and NP-specific antibodies as indicated in Materials and Methods.

FIG. 3.

Kinetics of protein synthesis in PA mutant virus-infected cells. Cultures of MDCK cells were infected at 10 PFU/cell and radiolabeled in vivo with [³⁵S]methionine-cysteine for 60 min at the times indicated (hours postinfection). After the radiolabeling pulse, total cell extracts were prepared and analyzed by polyacrylamide gel electrophoresis and autoradiography. Sizes are shown to the left in kilodaltons.

FIG. 4.

Kinetics of viral RNA accumulation. MDCK cells were infected at 10 PFU/ml with wild-type and PA mutant viruses. At the times indicated, cells were collected, total RNA was isolated, and the accumulation of the different viral RNA species of segment 7 was determined by real-time RT-PCR amplification, as described in Ma-terials and Methods. (A) mRNA. (B) Top, vRNA. Bottom, cRNA. RNA levels are indicated as the mean ± half of the standard deviation values from duplicates samples from at least seven independent experiments.

FIG. 5.

Pathogenicity of wild-type and mutant recombinant viruses in mice. A total of 5 × 10⁵ PFU of influenza virus A/Victoria/3/75 strain or recombinant (R) viruses corresponding to series A (A) and B (B) were inoculated intranasally into mice. Six mice were used for each condition, and every 2 or 3 days, two of them were sacrificed and viral titers in the lung extracts were measured. Left panels, body weight. Right panels, viral titers.