SUMOylation of matrix protein M1 and filamentous morphology collectively contribute to the replication and virulence of highly pathogenic H5N1 avian influenza viruses in mammals

The matrix protein (M1) of influenza A virus plays an important role in replication, assembly, and budding. A previous study found that aspartic acid (D) at position 30 and alanine (A) at position 215 of M1 contribute to the high pathogenicity of H5N1 viruses in mice, and double mutations of D to asparagine (N) at position 30 (D30N) and A to threonine (T) at position 215 (A215T) in M1 dramatically attenuate H5N1 viruses in mice. However, the underlying mechanisms by which these M1 mutations attenuate the virulence of H5N1 viruses are unknown. Here, we found that the amino acid mutation A215T eliminates the SUMOylation of M1 by reducing its interaction with the host SUMO1 protein, significantly reducing the stability of M1, slowing the export of the M1-vRNP complex from the nucleus to the cytoplasm, and reducing viral replication in MDCK cells. We further found that the D30N mutation in M1 alters the shape of progeny viruses from filamentous to spherical virions. Our findings reveal an essential role for M1 215A SUMOylation and M1 30D-related filamentous morphology in the pathogenesis of avian influenza viruses, which could be targeted in novel antiviral drug designs. Importance Identification of the pathogenic mechanism of highly pathogenic avian influenza viruses in mammals is helpful to develop novel anti-influenza virus strategies. Two amino acid mutations (D30N and A215T) in M1 were found to collectively attenuate H5N1 influenza viruses in mice, but the underlying mechanism remained unknown. This study found that the A215T mutation significantly decreases the SUMOylation of M1, which in turn attenuates the replication of H5N1 virus in mammalian cells. The D30N mutation in M1 was found to change the virion shape from filamentous to spherical. These findings are important for understanding the molecular mechanism of virulence of highly pathogenic avian influenza viruses in mammals.

An Intracellular Human Single Chain Antibody to Matrix Protein (M1) of H5N1 Virus

Background: Influenza virus matrix protein M1 is encoded by viral RNA fragment 7 and is the most abundant protein in virus particles. M1 is expressed in the late stages of viral replication and exerts functionality by inhibiting viral transcription. The M1 protein sequence is an attractive target for antibody drugs.Methods: The M1 protein sequence was amplified by RT-PCR using cDNA from the H5N1 virus as a template; the M1 protein was then expressed and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. The differences between HuScFv and TAT-HUScFv were verified by various experiments and the amino acid binding site of the M1 protein was determined.Results: The M1 protein of H5N1, HuScFv, and TAT-HuScFv, were successfully purified and expressed by and in E. coli. Further analysis demonstrated that TAT-HuScFv inhibited the hemagglutination activity of the 300TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.Conclusion: Recombinant HuScFv was successfully identified using the Tomlinson (I+J) phage antibody library and successfully linked to the TAT protein transductive domain of the HIV virus. Compared with the HuScFv, the addition of the TAT peptide improved its ability to penetrate the cell membrane. A definite amino acid binding site was identified after the decomposition of M1 protein, thus providing a target and reference for the development of antibody drugs and the study of new drugs.

Mass Spectrometric Characterization of Narcolepsy-Associated Pandemic 2009 Influenza Vaccines

The onset of narcolepsy, an irreversible sleep disorder, has been associated with 2009 influenza pandemic (pH1N1) infections in China, and with ASO3-adjuvanted pH1N1 vaccinations using Pandemrix in Europe. Intriguingly, however, the increased incidence was only observed following vaccination with Pandemrix but not Arepanrix in Canada. In this study, the mutational burden of actual vaccine lots of Pandemrix (n = 6) and Arepanrix (n = 5) sourced from Canada, and Northern Europe were characterized by mass spectrometry. The four most abundant influenza proteins across both vaccines were nucleoprotein NP, hemagglutinin HA, matrix protein M1, with the exception that Pandemrix harbored a significantly increased proportion of neuraminidase NA (7.5%) as compared to Arepanrix (2.6%). Most significantly, 17 motifs in HA, NP, and M1 harbored mutations, which significantly differed in Pandemrix versus Arepanrix. Among these, a 6-fold higher deamidation of HA146 (p.Asn146Asp) in Arepanrix was found relative to Pandemrix, while NP257 (p.Thr257Ala) and NP424 (p.Thr424Ile) were increased in Pandemrix. DQ0602 binding and tetramer analysis with mutated epitopes were conducted in Pandemrix-vaccinated cases versus controls but were unremarkable. Pandemrix harbored lower mutational burden than Arepanrix, indicating higher similarity to wild-type 2009 pH1N1, which could explain differences in narcolepsy susceptibility amongst the vaccines.

Mass Spectrometric Characterization of Narcolepsy-Associated Pandemic 2009 Influenza Vaccines

The onset of narcolepsy, an irreversible sleep disorder, has been associated with 2009 influenza pandemic (pH1N1) infections in China, and with ASO3-adjuvanted pH1N1 vaccinations using Pandemrix in Europe. Intriguingly, however, the increased incidence was only observed following vaccination with Pandemrix but not Arepanrix in Canada. In this study, the mutational burden of actual vaccine lots of Pandemrix (n=6) and Arepanrix (n=5) sourced from Canada, and Northern Europe were characterized by mass spectrometry. The four most abundant influenza proteins across both vaccines were nucleoprotein NP, hemagglutinin HA, matrix protein M1, with the exception that Pandemrix harbored a significantly increased proportion of neuraminidase NA (7.5%) as compared to Arepanrix (2.6%). Most significantly, 17 motifs in HA, NP, and M1 harbored mutations, which significantly differed in Pandemrix versus Arepanrix. Among these, a 6-fold higher deamidation of HA146 (N to D) in Arepanrix was found relative to Pandemrix, while NP257 (T to A) and NP424 (T to I) were increased in Pandemrix. DQ0602 binding and tetramer analysis with mutated epitopes were conducted in Pandemrix-vaccinated cases versus controls but were unremarkable. Pandemrix harbored lower mutational burden than Arepanrix, indicating higher similarity to wild-type 2009 pH1N1, which could explain differences in narcolepsy susceptibility amongst the vaccines.

The native structure of the full-length, assembled influenza A virus matrix protein, M1

Influenza A virus causes millions of severe illnesses during annual epidemics. The most abundant protein in influenza virions is the matrix protein M1 that mediates virus assembly by forming an endoskeleton beneath the virus membrane. The structure of full-length M1, and how it oligomerizes to mediate assembly of virions, is unknown. Here we have determined the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We found that the C-terminal domain of M1 is disordered in solution, but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerising M1 into linear strands which coat the interior surface of the assembling virion membrane. In the M1 polymer, five histidine residues, contributed by three different M1 monomers, form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore provide mechanisms for influenza virus assembly and disassembly.

Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization

The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1–M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains).