Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation

Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the host-cell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.