Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation

Abstract

The influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and ␤-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a "hot spot" to engineer ts mutants that could be used to design novel attenuated vaccines. IMPORTANCEBy targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number of ts mutations were engineered in such a short domain, demonstrating that rational design of ts mutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of these ts mutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs. Influenza A viruses (IAVs) are important viral respiratory pathogens of humans. These viruses are members of the Orthomyxoviridae family; they possess a negative-sense single-stranded segmented RNA genome (reviewed in reference 1). The three largest segments encode the three subunits of the RNA-dependent RNA polymerase: the two basic proteins PB1 and PB2 and the acidic subunit PA (reviewed in reference 2). In contrast to many RNA viruses, the influenza virus genome is transcribed and replicates in the nucleus of infected cells. The polymerase subuni...

Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

“…Fluorescently labeled PB1 and PA (wt and deletion mutant) subunits were first expressed separately in order to study their intracellular localization. Both subunits accumulated in the cytoplasm and did not migrate into the nucleus (not shown), in agreement with previous data (3,4,5,29). Next, we analyzed the localization of the PB1 subunit when coexpressed with wt PA or a PA codon deletion mutant (Fig.…”

“…In a recent study (29), we generated a series of temperature-sensitive mutants by single substitutions of proline for residues in a discrete domain (residues 210 to 226) of the polymerase PA subunit linker. These mutations were associated with a defect in the formation of the PA-PB1 complex and its import into the nucleus at a restrictive temperature (39.5°C).…”

“…During passage at 37°C, virus titers slowly declined, and assays aimed at isolating viruses that would be able to grow efficiently at 39.5°C failed. Thus, we were not able to select phenotypic revertants for the ⌬209 and ⌬210 virus mutants, while we were previously successful at rescuing (and genetically characterizing) revertants of proline substitution mutants in an adjacent stretch in the PA linker (29).…”

“…In a previous study (29), we showed that nucleotide substitutions generating proline codons in a PA linker domain resulted in the production of mutants that displayed a marked ts phenotype (Fig. 1B).…”

“…While several ts mutations have been identified in the polymerase subunits PA, PB1, and PB2 (23,24,25,26), what determines their ts phenotype often remains elusive. Several ts mutations in the PA linker resulting from nucleotide substitutions encoding proline were associated with a defect in polymerase complex assembly (27) and, more specifically, with an inability of the PA-PB1 dimer to associate efficiently with the importin IP05 (formerly named RanBP5 [28]) at a restrictive temperature, thus reducing its nuclear targeting (29). These ts mutations (as well as lethal mutations) are located in a helical segment (residues 209 to 218) and in a hairpin (residues 219 to 225) interacting with PB1.…”