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

Petra Švančarová,Tatiana Betáková

doi:10.1186/s12985-018-1056-x

Petra Švančarová, Tatiana Betáková

Open Access

https://doi.org/10.1186/s12985-018-1056-x

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Journal: Virology journal	Publication Date: Dec 1, 2018
Citations: 6	License type: open-access

Affiliation: Biomedicínske Centrum, Institute of Virology

Abstract

BackgroundThe 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.MethodsThe 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.ResultsMutant 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.ConclusionsOur finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.

Highlights

The influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection
M165 is essential for virus replication To test the role of single amino acids in the RQMV motif, we have generated plasmids with single mutation in Matrix protein (M1) gene
Since anti-M1 antibody did not work in western blot, we used anti-M2 polyclonal serum to compare expression of M2 protein after transfection with M1, R163A, Q164A, M165A, and V166A plasmids (Fig. 1a)

Summary

Introduction

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. The M1 protein is encoded by the M gene segment and comprised of 252 amino acids. This α-helical protein consists of two domains: N-terminal domain from 1 to 165 aa and C-terminal domain from 165 to 252 aa, which are linked by a protease sensitive loop. The N-terminal fragment has a globular structure and consists of nine α-helices and eight loop regions, and does not include any β-strands. N-domain and C-domain are connected by a loop region, which forms small α-helix (aa 162–166). The conservative MQMV motif presented in the α-helix possesses a glutamine-methionine cleavage site [10]

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