Fate of influenza A virus proteins

In this review we summarize the current state, history, future, mutation tendency and species susceptibility of influenza A virus proteins based on our probabilistic analyses on amino acid pairs, and compare the current state of influenza A virus proteins with that of proteins which we have studied in the past.     

Cellular immune responses of mice to influenza virus infection

Mice infected with influenza virus develop cytotoxic T lymphocytes (CTL) specific for viral antigens prior to the appearance of virus-specific antibody-forming cells (AFCs). Effector T cells were detected at a time coincident with a precipitous decline in pulmonary virus titer. CTLs of draining lymph nodes and spleen were found to be cross-reactive among H-2 compatible cells infected with influenza type A virus subtypes. AFCs were observed to be primarily hemagglutinin specific. Virus-specific IgA-secreting AFCs were detected in mediastinal lymph nodes of infected mice.     

Reevaluation of the proteins in rabies virus particles.

Protein content and localization of individual proteins of rabies virus have been studied. Four major proteins (estimated molecular weights, about 65,000, 54,000, 37,000 and 21,000), one minor component (molecular weight, about 200,000), and one intermediate (as regards its molar concentration) component (molecular weight, about 43,000) were revealed in rabies virus particles. In subviral particles accumulating in virus-infected cells, the 200,000-, 54,000-, and 37,000-dalton components were revealed. Some properties of the subviral particles allow them to be considered as viral nucleocapsids and the proteins composing them as analogs of L, N, and NS proteins of other rhabdoviruses. Thus, the protein composition of the rabies virus strain studied does not differ from that of other rhabdoviruses.     

Recognition of influenza virus proteins by cytotoxic T lymphocytes

Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro. Each peptide is recognized with a specific class I molecule (Db, Kk, Kd and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA-A2. The question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with non-viral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.     

Origin of small RNA in von Magnus particles of influenza virus.

A clone of recombinant virus obtained from the cross between WSN and Hong Kong strains of influenza virus gave rise to progeny containing predominantly von Magnus particles. In the electropherogram of virus RNA, the P3 gene was markedly diminished, and a new species of RNA (extra RNA) was present in addition to eight gene segments. The origin of the extra RNA was studied by two-dimensional gel electrophoresis of T1 RNase-generated oligonucleotides. Four out of five large oligonucleotide spots present in the extra RNA matched to those contained by the P3 gene. It was concluded that the extra RNA was derived from the P3 gene probably by deletion. The possible origin of the spot which was present in the extra RNA but not in eight gene segments including P3 was discussed.     

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.     

Annexin II incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin

For influenza viruses to become infectious, the proteolytic cleavage of hemagglutinin (HA) is essential. This usually is mediated by trypsin-like proteases in the respiratory tract. The binding of plasminogen to influenza virus A/WSN/33 leads to the cleavage of HA, a feature determining its pathogenicity and neurotropism in mice. Here, we demonstrate that plasminogen also promotes the replication of other influenza virus strains. The inhibition of the conversion of plasminogen into plasmin blocked influenza virus replication. Evidence is provided that the activation of plasminogen is mediated by the host cellular protein annexin II, which is incorporated into the virus particles. Indeed, the inhibition of plasminogen binding to annexin II by using a competitive inhibitor inhibits plasminogen activation into plasmin. Collectively, these results indicate that the annexin II-mediated activation of plasminogen supports the replication of influenza viruses, which may contribute to their pathogenicity.     

Cellular mechanisms involved in protection and recovery from influenza virus infection in immunodeficient mice.

We investigated the role of different lymphocyte subpopulations in the host defense reaction against influenza virus infection, taking advantage of various immunodeficient mouse strains. Whereas, following immunization, wild-type animals showed complete protection against challenge with a lethal dose of A/PR8/34 (PR8) virus, mice that lack both B and T cells but not NK cells (namely, scid and RAG2(-/-) mice) did not display any protective effect in similar conditions. By contrast, J(H)D(-/-) mice devoid of B cells and immunized with virus showed a protective response after challenge with a lethal dose. The immunized J(H)D(-/-) mice that survived completely recovered from the influenza virus infection. Immunized J(H)D(-/+) mice exhibited a more complete protection, suggesting the role of specific antibodies in resistance to infection. To assess the role of natural immunity in the host defense against influenza virus, we carried out experiments with scid mice challenged with lower but still lethal doses of PR8 virus. While an increased NK activity and an increased number of NK1.1+ cells in lungs of scid mice infected with PR8 virus were noted, in vivo depletion of the NK1.1+ cells did not affect the overall survival of the mice. Our results show that specific T cells mediate protection and recovery of J(H)D(-/-) mice immunized with live virus and challenged with lethal doses of influenza virus.     

Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Preparative isolation of basic structural proteins of the influenza virus]

The schemes for preparative electrophoretic isolation and purification of major proteins from influenza virus are described. The viral envelope protein, hemagglutinin, two of its subunits, internal M and NP proteins of influenza viruses A/FPV/Rostock (H7N1), A/PR/8/34 (H1N1) and X-31 (H3N2) were obtained in preparative amounts and characterized by amino acid and N-terminus analyses.     

Type I interferon antagonistic properties of influenza B virus polymerase proteins

The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections. In turn, many if not all viruses have evolved various means to circumvent, resist, or counteract this host response to ensure efficient replication and propagation. Influenza viruses are no exception to this rule, and several viral proteins have been described to possess IFN‐antagonistic functions. Although the viral nonstructural protein 1 appears to be a major antagonist in influenza A and B viruses (IAV and IBV), we have previously shown that a specific motif in the IAV polymerase proteins exerts an IFN‐suppressive function very early in infection. The question remained whether a similar function would also exist in IBV polymerases. Here, we show that indeed a specific amino acid position (A523) of the PB1 protein in the IBV polymerase complex confers IFN‐antagonistic properties. Mutation of this position leads to enhanced activation of the IFN‐mediated signalling pathway after infection and subsequent reduction of virus titres. This indicates that inhibition of innate immune responses is a conserved activity shared by polymerase proteins of IAV and IBV.