Influenza A Virus M1 Protein Non-Specifically Deforms Charged Lipid Membranes and Specifically Interacts with the Raft Boundary.

Topological rearrangements of biological membranes, such as fusion and fission, often require a sophisticated interplay between different proteins and cellular membranes. However, in the case of fusion proteins of enveloped viruses, even one molecule can execute membrane restructurings. Growing evidence indicates that matrix proteins of enveloped viruses can solely trigger the membrane bending required for another crucial step in virogenesis, the budding of progeny virions. For the case of the influenza A virus matrix protein M1, different studies report both in favor and against M1 being able to produce virus-like particles without other viral proteins. Here, we investigated the physicochemical mechanisms of M1 membrane activity on giant unilamellar vesicles of different lipid compositions using fluorescent confocal microscopy. We confirmed that M1 predominantly interacts electrostatically with the membrane, and its ability to deform the lipid bilayer is non-specific and typical for membrane-binding proteins and polypeptides. However, in the case of phase-separating membranes, M1 demonstrates a unique ability to induce macro-phase separation, probably due to the high affinity of M1's amphipathic helices to the raft boundary. Thus, we suggest that M1 is tailored to deform charged membranes with a specific activity in the case of phase-separating membranes.

Antigenic Architecture of the H7N2 Influenza Virus Hemagglutinin Belonging to the North American Lineage.

The North American low pathogenic H7N2 avian influenza A viruses, which lack the 220-loop in the hemagglutinin (HA), possess dual receptor specificity for avian- and human-like receptors. The purpose of this work was to determine which amino acid substitutions in HA affect viral antigenic and phenotypic properties that may be important for virus evolution. By obtaining escape mutants under the immune pressure of treatment with monoclonal antibodies, antigenically important amino acids were determined to be at positions 125, 135, 157, 160, 198, 200, and 275 (H3 numbering). These positions, except 125 and 275, surround the receptor binding site. The substitutions A135S and A135T led to the appearance of an N-glycosylation site at 133N, which reduced affinity for the avian-like receptor analog and weakened binding with tested monoclonal antibodies. Additionally, the A135S substitution is associated with the adaptation of avian viruses to mammals (cat, human, or mouse). The mutation A160V decreased virulence in mice and increased affinity for the human-type receptor analog. Conversely, substitution G198E, in combination with 157N or 160E, displayed reduced affinity for the human-type receptor analog.

Pleiotropic effects of hemagglutinin amino acid substitutions of influenza A(H1N1)pdm09 virus escape mutants.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We investigated pH optimum of fusion, temperatures of HA heat inactivation, in vivo and in vitro replication kinetics, and connectivity with panel of sera of survivors patients in different epidemic seasons of the previously obtained influenza H1 escape mutants. Our results showed that N133D (H3 numbering) mutation significantly lowered the pH of fusion optimum. Several amino acid substitutions, including K163 N, Q192 L, D190E, G228E, and K285 M, reduced the stability of HA as determined by heat inactivation, whereas A198E substitution is associated with significant increase in HA thermostability compared to the wild-type virus. We found that amino acid change D190 N was associated with a significant decrease in viral growth in eggs and mice. Our potential antigenic variants, except readapted variant, which contained A198E mutation, did not reach fixation in infected people. Overall, a co-variation between antigenic specificity and different HA phenotypic properties was demonstrated.

Effects of hemagglutinin amino acid substitutions in H9 influenza A virus escape mutants.

We assessed the pH optimum of fusion, HA thermostability, and in vitro replication kinetics of previously obtained influenza H9 escape mutants. The N198S mutation significantly increased the optimum pH of fusion. Four HA changes, S133N, T189A, N198D, and L226Q, were associated with a significant increase in HA thermostability compared to the wild-type virus. HA amino acid changes at positions 116, 133, 135, 157, 162, and 193 significantly decreased the replicative ability of H9 escape mutants in vitro. Monitoring of pleiotropic effects of the HA mutations found in H9 escape mutants is essential for accurate prediction of all possible outcomes of immune selection of H9 influenza A viruses.

Pleiotropic effects of amino acid substitutions in H5 hemagglutinin of influenza A escape mutants.

We believe that the monitoring of pleiotropic effects of the hemagglutinin (HA) mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. In the present study, we assessed multiple characteristics of antibody-selected HA mutations. We examined the pH optimum of fusion, HA heat inactivation, affinity to sialyl receptors, and in vitro and in vivo replication kinetics of various influenza H5 escape mutants. Several amino acid substitutions, including T108I, K152E, R162G, and K218N, reduced the stability of HA as determined by heat inactivation, whereas S128L and T215A substitutions were associated with significant increases in HA thermostability compared to the respective wild-type viruses. HA mutations at positions 108, 113, 115, 121, 123, 128, 162, and 190 and substitutions at positions 123, 199, and 215 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. The T108I substitution lowered the pH optimum of fusion and HA temperature stability while increasing viral replicative ability. Taken together, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated.

Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential.

Antigenic epitopes in the hemagglutinin of Qinghai-type influenza H5N1 virus.

The highly pathogenic avian influenza H5N1 viruses have become widespread and evolved into several clades. In our previous studies, the antigenic sites of the H5 hemagglutinin (HA) were characterized by selection and sequencing of escape mutants. In the present studies we analyzed the antigenic epitopes recognized by monoclonal antibodies against avian influenza A/Duck/Novosibirsk/56/05 (H5N1) virus isolated in western Siberia and belonging to subclade 2.2 of the H5N1 viruses. The analysis revealed several antigenically relevant positions of amino acid residues in the globular head of the HA not encountered earlier in the escape mutants of the H5 subtype. The newly recognized positions (113, 117, 118, 120, and 123, mature H5 numbering) are concentrated in an area adjacent to the region described in earlier studies as corresponding to site B in H3 HA, but extending far beyond this area. The amino acid positions recognized by the monoclonal antibodies against A/Duck/Novosibirsk/56/05 (H5N1) virus differ from the positions recognized by the monoclonal antibodies against H5N2 influenza viruses. The data suggest that the evolution of the HA of H5 avian influenza viruses is associated not only with the changes of antigenic epitopes recognized by antibodies, but also with a change in the dominance of the immunogenicity of different sites in the HA.

Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice.

Antigenic mapping of the haemagglutinin (HA) molecule of H5 and H9 influenza viruses by selecting escape mutants with monoclonal anti-HA antibodies and subjecting the selected viruses to immunological analysis and sequencing has previously been performed. The viruses used as wild-type strains were mouse-adapted variants of the original H5 and H9 isolates. Phenotypic characterization of the escape mutants revealed that the amino acid change in HA that conferred resistance to a monoclonal antibody was sometimes associated with additional effects, including decreased virulence for mice. In the present study, the low-virulence H5 and H9 escape mutants were readapted to mice. Analysis of the readapted variants revealed that the reacquisition of virulence was not necessarily achieved by reacquisition of the wild-type HA gene sequence, but was also associated either with the removal of a glycosylation site (the one acquired previously by the escape mutant) without the exact restoration of the initial wild-type amino acid sequence, or, for an H5 escape mutant that had no newly acquired glycosylation sites, with an additional amino acid change in a remote part of the HA molecule. The data suggest that such 'compensating' mutations, removing the damaging effects of antibody-selected amino acid changes, may be important in the course of influenza virus evolution.