GENETIC EVIDENCE FOR MATRIX-GLYCOPROTEINS INTERACTIONS OF THE HUMAN METAPNEUMOVIRUS

Human metapneumovirus (hMPV) is a pathogen that belongs to the Pneumovirinae subfamily, within the Paramyxoviridae family. hMPV causes upper and lower respiratory tract infections worldwide, resulting in mild to severe airways diseases. Assembly of paramyxoviruses is highly complex and involves multiple interactions among a variety of viral and cellular factors. The matrix (M) protein of paramyxoviruses is considered a `driving force` of virion assembly, since it interacts with several components of the virus particle, including the viral ribonucleoprotein complex, lipid membranes and viral glycoproteins. However, many details of hMPV assembly are unknown.

Mass spectrometry analyses of hMPV virions revealed that the N-terminus of the M protein is phosphorylated, at least in one of four adjacent putative phosphorylation sites (S3, Y4, T8 and Y9). Of these residues, S3 and Y4 are highly conserved among members of the Pneumovirinae subfamily. We mutated each of these two residues, and the adjacent conserved E2 residue, in the context of our improved reverse genetics system¹ for this virus. The resulting M mutants, harboring the E2D, S3E and Y4F mutations, were all replication-competent but spread-defective. We screened for revertants of the S3E and Y4F clones, which their spreading was rescued by second-site suppressor mutations. Sequence analyses of such revertants revealed mutations in two of the three viral glycoproteins: small hydrophobic (SH) and glycoprotein (G), providing genetic evidence for M-SH and M-G interactions, which also may involve matrix phosphorylation. We will provide a model for these interactions, based on the recent published structure of hMPV M protein.

¹Sabo Y, et al., J Virol. 2011, 85(13):6594.