Conserved methionine 165 of matrix protein contributes to the nuclear import and is essential for influenza A virus replication

Background: The influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection. N-domain and C-domain are connected by a loop region, which consists of conserved RQMV motif. Methods: The function of the highly conserve RQMV motif in the influenza virus life cycle was investigated by site-directed mutagenesis and by rescuing mutant viruses by reverse genetics. Co-localization of M1 with nucleoprotein (NP), clustered mitochondria homolog protein (CLUH), chromosome region maintenance 1 protein (CRM1), or plasma membrane were studied by confocal microscopy. Results: Mutant viruses containing an alanine substitution of R163, Q164 and V166 result in the production of the virus indistinguishable from the wild type phenotype. Single M165A substitution was lethal for rescuing infection virus and had a striking effect on the distribution of M1 and NP proteins. We have observed statistically significant reduction in distribution of both M165A (p<0,05) and NP (p<0,001) proteins to the nucleus in the cells transfected with the reverse-genetic system with mutated M1. M165A protein was co-localized with CLUH protein in the cytoplasm and around the nucleus but transport of M165-CLUH complex through the nuclear membrane was restricted. Conclusions: Our finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.