Search

Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively. Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids. All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1-113 of the PB2 protein. Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates. The application of these antibodies as research and diagnostic tools is discussed.

Two panels of monoclonal antibodies (MAbs) specific for the influenza A virus PA and PB2 polypeptides have been obtained from mice immunized with denatured proteins produced in Escherichia coli. All MAbs (13 specific for the PA polypeptide and 8 specific for the PB2 protein) reacted to the corresponding influenza virus protein in Western blotting (immunoblotting), immunoprecipitation, and immunofluorescence assays. To gain information about the roles of the nucleoprotein (NP) and PB2 and PA proteins during viral mRNA synthesis, the 21 anti-P antibodies and 3 anti-NP antibodies (J. A. López, M. Guillen, A. Sánchez-Fauquier, and J. A. Melero, J. Virol. Methods 13:255-264, 1986) were purified and tested for their ability to inhibit the transcriptase activity associated with viral cores purified from virions. Four of the antibodies (one anti-PB2 and the three anti-NP MAbs) inhibited transcription by more than 50% compared with unrelated control antibodies. The inhibitory effect was not due to a nonspecific effect of the antibody preparations, because these MAbs did not inhibit transcription when tested on influenza B virus nucleocapsids, which are not recognized by the antibodies. To determine whether the antibodies were acting on an early transcription step, transcription reactions were carried out in the presence of globin mRNA (a mixture of alpha- and beta-globin chains) and only one labeled nucleoside triphosphate (either GTP or CTP). The results obtained showed that MAbs to the PB2 and NP polypeptides interfered with the initiation step of mRNA-primed transcription. The implications of these results regarding initiation of viral mRNA synthesis are discussed.

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.

BACE2 is homologous to BACE1, a beta-secretase that is involved in the amyloidogenic pathway of amyloid precursor protein (APP), and maps to the Down syndrome critical region of chromosome 21. Alzheimer disease neuropathology is common in Down syndrome patients at relatively early ages, and it has thus been speculated that BACE2 co-overexpression with APP would promote the early neurodegenerative phenotype. However, the in vivo function of BACE2 has not yet been elucidated. The aim of the present work has been to analyse the impact of in vivo BACE2 overexpression using a transgenic mouse model. Our results suggest that BACE2 is not involved in the amyloidogenic pathway, cognitive dysfunction or cholinergic degeneration. However, TgBACE2 animals showed increased anxiety-like behaviour along with increased numbers of noradrenergic neurones in locus coeruleus, thus suggesting an unexpected role of BACE2 overexpression.