Specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses

Abstract

Human metapneumovirus (HMPV) and avian metapneumovirus (AMPV) have a similar genome organization and protein composition, but a different host range. AMPV subgroup C (AMPV-C) is more closely related to HMPV than other AMPVs. To investigate the specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses, a minireplicon system was generated for AMPV-C and used in combination with minireplicon systems for HMPV lineages A1 and B1. Viral RNA-like molecules representing HMPV-A1 and -B1, AMPV-A and -C and human respiratory syncytial virus were replicated efficiently by polymerase complexes of HMPV-A1 and -B1 and AMPV-C, but not by polymerase complexes of bovine parainfluenza virus 3. Upon exchange of HMPV and AMPV-C polymerase complex components, all chimeric polymerase complexes were functional; exchange between HMPVs did not result in altered polymerase activity, whereas exchange between HMPVs and AMPV-C did. Recombinant HMPV-B1 viruses in which polymerase genes were exchanged with those of HMPV-A1 replicated with normal kinetics in vitro, whilst replacement with AMPV-C genes resulted in moderate differences in virus replication. In hamsters, recombinant HMPV-B1 viruses in which individual polymerase genes were exchanged with those of AMPV-C were attenuated, irrespective of the results obtained with minireplicon systems or in vitro replication assays. This study provides insight into the specificity and functional interaction of polymerase complex proteins of human and avian metapneumoviruses, but neither minireplicon systems nor in vitro replication kinetics were found to be predictive for attenuation in permissive animals. INTRODUCTIONHuman metapneumovirus (HMPV) is an enveloped, nonsegmented, negative-strand RNA virus that causes respiratory tract illnesses, primarily in infants, young children, the frail elderly, and immunocompromised individuals (Crowe, 2004;Falsey et al., 2003;Kahn, 2006;Pelletier et al., 2002;van den Hoogen et al., 2001 van den Hoogen et al., , 2003. HMPV is a member of the family Paramyxoviridae, subfamily Pneumovirinae, genus Metapneumovirus, and can be divided in two main genetic lineages (A and B) each consisting of two sublineages, A1 and A2, and B1 and B2 (van den Hoogen et al., 2004). The only other member of the genus Metapneumovirus is avian metapneumovirus (AMPV). AMPV has been found to infect domestic poultry worldwide, causing acute respiratory infections (Cook, 2000). AMPVs have been classified into four subgroups, A-D (Bayon-Auboyer et al., 1999;Eterradossi et al., 1995;Juhasz & Easton, 1994;Seal, 1998). AMPV-C was first detected in the USA and is more closely related to HMPV than the other AMPV subgroups (Govindarajan & Samal, 2004Govindarajan et al., 2004;Toquin et al., 2003;van den Hoogen et al., 2002;Yunus et al., 2003). Human respiratory syncytial virus (HRSV) is the only other member of the subfamily Pneumovirinae that infects humans. In comparison with HRSV, metapneumoviruses lack the non-structural proteins NS1 and NS2,...

Evolutionary dynamics of human and avian metapneumoviruses

“…Finally, AMPV subgroup C was first detected in the USA in 1996 and is more closely related to HMPV than to any other AMPV subgroup (Govindarajan & Samal, 2004Govindarajan et al, 2004;Toquin et al, 2003; ). However, using both chimeric viruses and minireplicon systems it has been shown that the ribonucleoprotein complex proteins of HMPV and AMPV-C are functionally interchangeable (de Graaf et al, 2008;Govindarajan et al, 2006;Pham et al, 2005). Spill-over events of pathogens from birds to humans are not uncommon (Reed et al, 2003).…”

Fusion protein is the main determinant of metapneumovirus host tropism

“…In contrast to the avian cell lines, Vero-118 cells supported efficient replication of both HMPV and AMPV-C (Fig. 3) (de Graaf et al, 2008a).…”

“…Genome replication requires the viral genomic RNA (vRNA) to be encapsidated in a ribonucleoprotein complex, comprising the nucleoprotein (N), which encapsidates the viral genome, the phosphoprotein (P), the large polymerase protein (L) and the putative transcriptional elongation factor (M2.1). These polymerase complex proteins can be functionally exchanged between HMPV and AMPV-C in both mini-replicon systems and chimeric viruses (de Graaf et al, 2008a;Govindarajan et al, 2006;Pham et al, 2005). Metapneumoviruses encode three membrane glycoproteins, the attachment (G), the small hydrophobic (SH) and the fusion (F) proteins.…”

“…The full-length cDNA plasmids for HMPV (lineage B1, strain NL/1/99) and the full-length chimeric HMPV cDNA plasmids containing the N, P or L gene of AMPV-C have been described previously (de Graaf et al, 2008a;Herfst et al, 2004). For construction of the full-length chimeric HMPV cDNA plasmids containing the F, G or SH gene of AMPV-C, fragments of HMPV were amplified by PCR and cloned into pBluescript SK + or pCR4TOPO (Invitrogen).…”

“…Each day, 0.5 ml supernatant was collected and replaced by fresh medium. Plaque assays were performed in Vero-118 cells to determine virus titres as described previously (de Graaf et al, 2008a).…”