Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.     

Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.     

Detection of influenza virus nucleoprotein on the surface of infected cells and in free nonvirion form

The inner viral nucleoprotein synthesized de novo is shown to be exposed on the surface of the chicken embryo infected with influenza virus. The kinetics of the nucleoprotein located on the surface does not correlate with the kinetics of cell destruction. In culture or allantois virus containing liquids the large number of extracellular viral nucleoprotein prone to antinucleoprotein monoclonal antibodies was found. The accumulation of this nucleoprotein occurs in the period when cell destruction is absent, it is eliminated by the adsorption of the virus on erythrocytes or centrifugation at 70,000 g for 2 hours (20%) or by centrifugation at 10,000 g for 4 hours (50%).     

Influenza Nucleoprotein Delivered with Aluminium Salts Protects Mice from an Influenza A Virus That Expresses an Altered Nucleoprotein Sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.     

Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2

An influenza B virus nucleoprotein (BNP) peptide, residues 82-94, defined by limited sequence homology with an HLA-A2-restricted peptide from influenza A matrix protein, was recognized by HLA-A2-restricted CTL. Reciprocal inhibition of T cell recognition by the two peptides suggest that the BNP peptide may have lower avidity for HLA-A2 molecules than the matrix peptide. The interaction between this peptide and HLA-A2 was explored by studying the CTL recognition of BNP 82-94 presented by mutant HLA-A2 molecules. Mutations at residues 9, 99, 70, 74, 152 and 156 were found to abolish T cell recognition of the BNP peptide. These results were compared with results previously obtained with the influenza A matrix peptide and suggest that the two peptides bind differently in the peptide binding site.     

Functional compensation of a detrimental amino acid substitution in a cytotoxic-T-lymphocyte epitope of influenza a viruses by comutations

Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs.     

Determination of influenza virus proteins required for genome replication.

An artificial vaccinia virus vector-driven replication system for influenza virus RNA has been developed. In this system, a synthetic NS-like gene is replicated and expressed by influenza virus proteins supplied through infection with vaccinia virus recombinant vectors. The minimum subset of influenza virus proteins needed for specific replication and expression of the viral ribonucleoprotein was found to be the three polymerase proteins (PB1, PB2, and PA) and the nucleoprotein.     

Granzyme K inhibits replication of influenza virus through cleaving the nuclear transport complex importin α1/β dimer of infected host cells

The influenza A virus is a causative agent of influenza, which infects human cells and uses host factors to accomplish viral genome replication as part of its life cycle. The nucleoprotein (NP) and PB2 of the influenza virus associate with importin α1 to gain access to the host nucleus through a ternary import complex. Killer cell-mediated cytotoxicity is the primary mechanism of eliminating the influenza virus. Here, we showed that lymphokine-activated killer cells participated in the elimination of the influenza virus. Granzyme (Gzm) K inhibition elevated viral replication in vitro and aggravated viral infection in vivo. We identified that importin α1 and its transport partner protein importin β are physiological substrates of GzmK. Proteolysis of these two substrates wrecked their association to generate the importin α1/β dimer and disrupted transportation of viral NP to the nucleus, leading to inhibition of influenza virus replication.     

Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes.

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.     

Three-dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle

A three-dimensional structural model of an influenza virus ribonucleoprotein particle reconstituted in vivo from recombinant proteins and a model genomic vRNA has been generated by electron microscopy. It shows a circular shape and contains nine nucleoprotein monomers, two of which are connected with the polymerase complex. The nucleoprotein monomers show a curvature that may be responsible for the formation of helical structures in the full-size viral ribonucleoproteins. The monomers show distinct contact boundaries at the two sides of the particle, suggesting that the genomic RNA may be located in association with the nucleoprotein at the base of the ribonucleoprotein complex. Sections of the three-dimensional model show a trilobular morphology in the polymerase complex that is consistent with the presence of its three subunits.     

Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.