Antigenic reactivity and electrophoretic migrational heterogeneity of the three polymerase proteins of type A human and animal influenza viruses

Summary Antigenic reactivity of the three polymerase proteins PB1, PB2, and PA of type A influenza viruses of animal and human origin were analysed by radioimmunoprecipitation using monospecific antisera. Each of the polymerase monospecific antisera made against the polymerase proteins of the human A/WSN/33 (H1N1) influenza virus reacted efficiently with the homologous proteins of all the known thirteen HA subtype viruses of avian influenza virus, three subtypes of human influenza virus, swine and equine influenza viruses. This broad reactivity of each of the antisera indicated that the polymerase proteins are antigenically related among the type A influenza viruses and therefore can be considered as type specific antigens similar to the other viral internal proteins nucleoprotein (NP) and matrix protein (M). No electrophoretic migrational heterogeneity was found among the PB2 proteins of different subtype viruses, whereas PB1 protein exhibited minor variation. However, PA protein from among various viral subtypes showed considerable heterogeneity. Each of the polymerase antisera also immunoprecipitated additional antigenically related polypeptides with distinct electrophoretic mobilities from cells infected with each of the influenza viral subtypes.     

Hsp90 inhibitors reduce influenza virus replication in cell culture

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.     

Influenza a virus PB1-F2 protein

PB1-F2 protein (PB1-F2) is encoded by the alternative (+1) ORF in the PB1 gene of influenza A viruses (IAVs). This protein has a number of unique features, namely its absence from some animal IAV isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation, mitochondrial localization, and apoptotic or pro-apoptotic properties. Localization of PB1-F2 to mitochondria is mediated via C-terminal basic amphipathic alpha-helix. PB1-F2 affects apoptosis and may contribute to the pathogenicity and lethality of IAVs. Sequence analysis showed that, in addition to the strains with an ORF for full-length PB1-F2, there are some with an ORF for different truncated forms of PB1-F2. Several other viruses encode proteins with structure and function similar to PB1-F2 of IAVs.     

Nuclear transport of influenza virus polymerase PA protein.

The subcellular distribution of influenza polymerase PA subunit has been studied using a SV40-recombinant virus (SVPA76), which allows the expression and accumulation of this protein in COS-1 cells. In contrast to the complete nuclear localization observed for the PA subunit several hours after influenza virus infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA protein accumulated either in the nucleus, in the cytoplasm or was distributed throughout the cell. When cells were infected with the SVPA76 recombinant and superinfected with influenza virus, a clear increase in the proportion of cells showing nuclear localization of the PA protein was observed, suggesting that some trans-factor may be required to allow complete nuclear accumulation of the protein. Double infections using SVPA76 recombinant and either SVPB1 or SVNS recombinant viruses showed a complete correlation between expression of polymerase PB1 subunit or NS1 protein and nuclear localization of polymerase PA subunit. However, no such correlation was observed in the double infections of SVPA76 and SVNP recombinants. These results suggest that polymerase PB1 subunit and the non-structural proteins could be involved in the nuclear targeting or nuclear retention of influenza polymerase PA protein.     

Avian cells expressing the murine Mx1 protein are resistant to influenza virus infection

The cDNA encoding the murine Mx1 protein, a mediator of resistance to influenza virus, was inserted into a replication-competent avian retroviral vector in either the sense (referred to as Mx+) or the antisense (referred to as Mx-) orientation relative to the viral structural genes. Both vectors produced virus retaining the Mx insert (Mx recombinant viruses referred to as Mx+ and Mx-) following transfection into chicken embryo fibroblasts (CEF). Mx protein of the appropriate size and nuclear localization was expressed only in CEF cells infected with the Mx+ virus. Mx expression was observed in all Mx(+)-infected cells and was stable during long-term culture. Cells infected with the Mx+ virus were resistant to infection by human influenza A/WSN/33 (H1N1) and avian influenza viruses A/Turkey/Wisconsin/68 (H5N9) and A/Turkey/Massachusetts/65 (H6N2), but were susceptible to infection by the enveloped RNA viruses Sindbis and vesicular stomatitis virus (VSV). Normal CEF and cells infected with the Mx virus were susceptible to influenza A, Sindbis, and VSV. The synthesis of influenza proteins, especially the larger polymerase and hemagglutinin proteins, was reduced in Mx+ retrovirus-infected cells superinfected by influenza A.     

Single mutation at the amino acid position 627 of PB2 that leads to increased virulence of an H5N1 avian influenza virus during adaptation in mice can be compensated by multiple mutations at other sites of PB2

To understand the adaptation of H5N1 influenza viruses to mammals, a non-pathogenic influenza H5N1 virus (HN021) in mice was passaged for 15 times in mammalian host. Animal experimental results indicated that the mouse-adapted (MA) variants became highly pathogenic in mice after the passages. Sequence analysis showed that there was one amino acid substitution in PB2 protein of MA mutants after first passage (MA1), three amino acid substitutions in PB2 protein of MA5 and one amino acid in M1 protein, seven amino acids in HA protein and seven amino acids in PB2 protein of MA15, respectively. Animal experiments and growth assays with reassortant viruses produced by reverse genetics showed that mutations in PB2 alone contributed to the increase in virulence of HN021 in mice. Polymerase activity assays showed that the mutations in PB2 enhanced ribonucleoprotein complex polymerase activity in mammalian cells. Interestingly, one reverse mutation (K627E) took place at the amino acid position 627 of PB2 during passages of MA5 to MA15, indicating that a lysine at position 627 of PB2 is not absolutely needed for virulence and adaptation in mice by H5N1 virus. Taken together, the results suggest that mutations at multiple sites of PB2 contributed to the virulence and adaptation in mice, and the E627K mutation of PB2 is not an indispensable determinant in PB2 for mammalian adaptation by H5N1 avian influenza virus.     

Comparison of the pathogenesis of two genetically different H3N2 influenza A viruses in pigs

In 1997 and 1998, H3N2 influenza A viruses emerged among pigs in North America. Genetic analyses of the H3N2 isolates demonstrated that they had distinctly different genotypes. The most commonly isolated viruses in the United States have a triple-reassortant genotype, with the hemagglutinin, neuraminidase, and PB1 polymerase genes being of human influenza virus origin, the nucleoprotein, matrix, and nonstructural genes being of classical swine influenza virus origin, and the PA and PB2 polymerase genes being of avian influenza virus origin. In contrast, a wholly human H3N2 virus was isolated from a single baby pig in Ontario, Canada, in 1997, but it did not spread within the swine population. Genetic differences between this wholly human virus and the triple-reassortant viruses may affect their replication efficiencies in pigs. In the present study we compared the pathogenicities and replication kinetics of the wholly human virus and a triple-reassortant virus in 7-week-old pigs that were infected intranasally with 2 x 10(3) to 2 x 10(6) 50% tissue culture infective doses of virus. Our results demonstrate that the wholly human virus replicated to significantly lower titers and that the onset of virus shedding was delayed compared to the replication titers and the time of onset of virus shedding in triple-reassortant viruses. In addition, infection with the triple-reassortant virus was associated with moderate to severe gross pathological and histological pulmonary lesions, while infection with the wholly human virus induced only mild pulmonary changes.     

Comparison of the three large polymerase proteins of influenza A, B, and C viruses

The three large RNA segments of influenza C virus C/JJ/50 were cloned and sequenced, and the deduced amino acid sequences were compared with those of the polymerase (P) proteins of influenza A and B viruses. The coding strategy of the C virus RNA segments is the same as that for the large A and B virus segments as one long open reading frame is present in each segment. RNA segment 1 of influenza C virus encodes the equivalent of the PB2 protein; it has an approximate 25% sequence identity with the corresponding (cap binding) influenza A and B virus PB2 proteins. The PB1 protein of influenza C virus, coded for by segment 2, has an approximate 40% sequence identity with the corresponding proteins of influenza A and B viruses including the Asp-Asp sequence motif found in many RNA polymerase molecules. The PB1 polymerase is thus the most highly conserved protein among the influenza A, B, and C viruses. Although the protein coded for by RNA 3 of influenza C virus shows an approximate 25% sequence identity with the acid polymerase (PA) proteins of the A and B viruses, its sequence does not display any acid charge features at neutral pH. This protein is thus referred to as the P3 (rather than the PA) protein of influenza C virus.     

Both influenza hemagglutinin and polymerase acidic genes are important for delayed pandemic 2009 H1N1 virus clearance in the ferret model

We previously showed that a pandemic virus, A/Tennessee/560/09(H1N1), had the potential to adapt to human bronchial epithelial cells by the acquisition of hemagglutinin (HA) K154Q and polymerase acidic (PA) protein L295P mutations that conferred a more virulent phenotype. To better elucidate the role of each mutations, we generated recombinant viruses carrying single mutations or both mutations concurrently. The replication of all mutant viruses was significantly higher than that of the wild-type A/Tennessee/560/09 virus in human cells. The HA K154Q mutation reduced the receptor binding affinity of A/Tennessee/560/09 virus to 6-Su-6′SLN and biantennary 6′SLN receptors. In ferrets, H1N1 virus with HA K154Q and PA L295P mutations exhibited significantly higher titers in the upper respiratory tract compared to all other viruses 6 days post-infection. Our results suggest that both single mutations HA K154Q and PA L295P are necessary for delayed virus clearance of A/Tennessee/560/09(H1N1) influenza virus in a ferret animal model.     

Three amino acid changes in PB1-F2 of highly pathogenic H5N1 avian influenza virus affect pathogenicity in mallard ducks

Despite reports that the PB1-F2 protein contributes to influenza virus pathogenicity in the mouse model, little is known about its significance in avian hosts. In our previous study, the A/Vietnam/1203/04 (H5N1) wild-type virus (wtVN1203) was more lethal to mallard ducks than a reverse genetics (rg)-derived VN1203. In search of potential viral factors responsible for this discrepancy, we found that synonymous mutations (SMs) had been inadvertently introduced into three genes of the rgVN1203 (rgVN1203/SM-3). Of 11 SMs in the PB1 gene, three resided in the PB1-F2 open reading frame, caused amino acid (aa) substitutions in the PB1-F2 protein, and reduced virus lethality in mallard ducks. The wtVN1203 and recombinant viruses with repairs to these three aa’s (rgVN1203/R-PB1-F2) or with repairs to all 11 SMs (rgVN1203/R-PB1) were significantly more pathogenic than rgVN1203/SM-3. In cultured cells, repairing three mutations in PB1-F2 increased viral polymerase activity and expression levels of viral RNA.     

Enhanced infectivity of H5N1 highly pathogenic avian influenza (HPAI) virus in pig ex vivo respiratory tract organ cultures following adaptation by in vitro passage

Pigs are thought to play a role in the adaptation of avian influenza (AI) viruses to mammalian hosts. To better understand this mechanism and to identify key mutations two highly pathogenic AI (HPAI) viruses (H5N1 and H7N7) were grown in pig cells, To mimic the pressure of an immune response, these viruses were grown in the presence of antiserum to the homologous virus or porcine IFN-γ. Mutations were identified in both viruses grown in vitro in the presence and absence of antisera or IFN-γ and included the PB2 mutations, E627K or 627E,D701N, described previously as requirements for the adaptation of AI viruses to mammalian species. Additional mutations were also identified in PB1, HA, NP and M genes for viruses passaged in the presence of immune pressure. The infectivity of these viruses was then assessed using ex vivo pig bronchi and lung organ cultures. For lung explants, higher levels of virus were detected in organ cultures infected with H5N1 HPAI viruses passaged in pig cell lines regardless of the presence or absence of homologous antisera or IFN-γ when compared with the wild-type parental viruses. No infection was observed for any of the H7N7 HPAI viruses. These results suggest that the mutations identified in H5N1 HPAI viruses may provide a replication or infection advantage in pigs in vivo and that pigs may continue to play an important role in the ecology of influenza A viruses including those of avian origin.     

Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses

A complete set of recombinant vaccinia viruses that express each of the influenza virus polypeptides has been constructed. PB1, PB2, PA, HA, NP, M1, and NS1 genes were derived from influenza virus A/PR/8/34, NA from influenza virus A/Cam/46, and M2 and NS2 genes from influenza virus A/Udorn/72. Cells infected with these recombinant viruses synthesize influenza polypeptides that are precipitable with specific antisera and that have electrophoretic mobilities similar to the corresponding influenza virus polypeptides. Indirect immunofluorescence studies have shown that HA, NA, and MS2 proteins migrate to the cell surface; PB2, PB1, PA, NP, and NS1 proteins migrate to the cell nucleus; and M1 and NS2 are distributed throughout the cell, although NS2 accumulates preferentially in nuclei. These transport processes occurred independently of other influenza polypeptides and are therefore attributable to the intrinsic properties of the influenza polypeptides themselves.     

Nucleotide sequence of the avian influenza A/Mallard/NY/6750/78 virus polymerase genes

The avian influenza A/Mallard/NY/6750/78 virus is currently being evaluated as a donor of attenuating genes in the construction of live avian-human influenza A reassortant virus vaccines for use in humans. We determined the nucleotide sequences of the three polymerase gene segments of this virus. This completes the nucleotide sequence of the six transferrable genes of the avian donor virus. Comparison of the nucleotide and deduced amino acid sequences of the non-glycoprotein genes of the avian A/Mallard/78 virus with representative avian and human influenza A viruses suggests that the PB1 gene of H2N2 subtype human influenza A viruses may have been derived from a non-human, possibly avian influenza A virus by genetic reassortment. In addition, several regions of conserved amino acids with potential functional significance were identified in the deduced amino acid sequences of the polymerase proteins.     

Evolutionary relatedness of the predicted gene product of RNA segment 2 of the tick-borne Dhori virus and the PB1 polymerase gene of influenza viruses

The complete nucleotide sequence of the second largest RNA segment of Dhori/India/1313/61 virus was determined and the deduced amino acid sequence was compared with the polymerase (P) proteins of influenza A, B, and C viruses. RNA segment 2 (2224 nucleotides) of Dhori virus contains a single long open reading frame that can encode a 716-amino acid polypeptide (81.3 kDa). The predicted polypeptide shares between 27 and 31% sequence identities with the PB1 polypeptides of influenza A, B, and C viruses. Among the regions most highly conserved are the sequences around the Asp-Asp motif common to many RNA polymerases. In spite of the high level of sequence identity between the Dhori RNA segment 2 gene product and the influenza A, B, and C virus PB1 proteins the amino acid composition of the Dhori protein indicates an acidic charge feature at pH 7.0 in contrast to the basic nature of the PB1 proteins of the influenza viruses. We suggest that the Dhori PB1-like protein be designated the P alpha protein of this virus.     

Factors influencing helper-independent adeno-associated virus replication

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.