The influenza virus RNA-dependent RNA polymerase which is composed of three subunits, PB1, PB2, and PA, catalyses genome replication and transcription within the cell nucleus. The PA linker (residues 197-256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts) and attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for further in vitro and in vivo characterization. Four single-codon deletion viable mutants were generated; all of them exhibited a ts-phenotype that was associated to a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at restrictive temperature (39.5°C). Mice infections showed that the four mutants were attenuated and induced antibodies able to protect from a lethal homologous wild-type virus challenge. Serial in vitro passages of two deletion mutants at 39.5°C and 37°C did not allowed to restore a wt-phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stable ts-mutants that could be used to design novel attenuated vaccines.