Identification of a truncated nucleoprotein in avian metapneumovirus-infected cells encoded by a second AUG, in-frame to the full-length gene

Abstract

Background: Avian metapneumoviruses (aMPV) cause an upper respiratory disease with low mortality, but high morbidity primarily in commercial turkeys. There are three types of aMPV (A, B, C) of which the C type is found only in the United States. Viruses related to aMPV include human, bovine, ovine, and caprine respiratory syncytial viruses and pneumonia virus of mice, as well as the recently identified human metapneumovirus (hMPV). The aMPV and hMPV have become the type viruses of a new genus within the Metapneumovirus. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. Based on predicted antigenicity of consensus protein sequences, five aMPV-specific N peptides were synthesized for development of peptide-antigens and antisera.

Results: The presence of two aMPV nucleoprotein (N) gene encoded polypeptides was detected in aMPV/C/US/Co and aMPV/A/UK/3b infected Vero cells. Nucleoprotein 1 (N1) encoded from the first open reading frame (ORF) was predicted to be 394 amino acids in length for aMPV/C/US/Co and 391 amino acids in length for aMPV/A/UK/3b with approximate molecular weights of 43.3 kilodaltons and 42.7 kilodaltons, respectively. Nucleoprotein 2 (N2) was hypothesized to be encoded by a second downstream ORF in-frame with ORF1 and encoded a protein predicted to contain 328 amino acids for aMPV/C/US/Co or 259 amino acids for aMPV/A/UK/3b with approximate molecular weights of 36 kilodaltons and 28.3 kilodaltons, respectively. Peptide antibodies to the N-terminal and C-terminal portions of the aMPV N protein confirmed presence of these products in both aMPV/C/US/Co- and aMPV/A/UK/3b-infected Vero cells. N1 and N2 for aMPV/C/US/Co ORFs were molecularly cloned and expressed in Vero cells utilizing eukaryotic expression vectors to confirm identity of the aMPV encoded proteins.

Conclusion: This is the first reported identification of potential, accessory in-frame N2 ORF gene products among members of the Paramyxoviridae. Genomic sequence analyses of related members of the Pneumovirinae other than aMPV, including human respiratory syncytial virus and bovine respiratory syncytial virus demonstrated the presence of this second potential ORF among these agents.

Figure 1

Alignment of avian metapneumovirus type A and C nucleoprotein genes demonstrating presence of multiple start sites. Underlined sequences denote hypothesized alternative in-frame start sites and the stop codon. Primer sequences utilized for cDNA synthesis of nucleoprotein genes are also illustrated.

Figure 2

Relative position of peptides within the avian metapneumovirus nucleoproteins utilized for generation of affinity purified polyclonal antibodies.

Figure 3

Detection of avian metapneumovirus (aMPV) nucleoprotein gene products among infected cells utilizing affinity purified peptide antibodies. A. Antibody reacted against an N-terminal portion of the nucleoprotein designed to detect all aMPV serotypes N1. Lane 1: molecular size markers; Lane 2: uninfected cell proteins; Lane 3: aMPV/A infected cell proteins; Lane 4: aMPV/B infected cell proteins; Lane 5: aMPV/C infected cell proteins. B. Antibody detection of a C-terminal portion of the aMPV/C nucleoprotein. Lane 1: uninfected cell proteins; Lane 2: aMPV/C infected cell proteins reacted with N1 peptide antibodies; Lane 3: aMPV/C infected cells reacted with aMPV/C-specific N2 peptide antibodies. C. Antibody detection of a C-terminal portion of the aMPV/A nucleoprotein. Lane 1: uninfected cell proteins; Lane 2: aMPV/A infected cell proteins reacted with N1 peptide antibodies; Lane 3: aMPV/A infected cell proteins reacted with N3 peptide antibodies; Lane 4: aMPV/A infected cells reacted with N5 peptide antibodies.

Figure 4

Expression of N1 and N2 open reading frames of avian metapneumovirus type C in transfected eukaryotic cells by an expression vector. Lane 1: molecular size markers; Lane 2: uninfected control cells; Lane 3. aMPV/C infected cells reacted with antibodies to peptide N1. Lane 4: Cells transformed with aMPV/C-N gene complete ORF reacted with antibodies to peptide N1. Lane 5: Cells transformed with expression plasmid with truncated N2ORF reacted to antibodies to peptide N1; Lane 6: uninfected control cells; Lane 7: aMPV/C infected cells reacted to antibodies to peptide N4. Lane 8: Cells transformed with aMPV/C-N gene complete ORF reacted with antibodies to peptide N2. Lane 9: Cells transformed with expression plasmid with truncated N2ORF reacted to antibodies to peptide N2.