An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters

Abstract

Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F 0 ) into F 1 and F 2 exposes a fusion peptide at the N terminus of the F 1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/ NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.Human metapneumovirus (hMPV) is a recently identified respiratory virus that was initially isolated from children in The Netherlands experiencing symptoms of acute respiratory disease with undetermined etiology. hMPV causes respiratory illness ranging from mild upper respiratory symptoms to severe lower respiratory disease such as bronchiolitis and pneumonia (6,60,63). Depending on the patient population sampled, between 5 and 15% of respiratory infections in young children may be attributable to hMPV infection (7,62,65). hMPV is also associated with 12 to 50% of otitis media in children (7,36,62). In The Netherlands, 55% of tested individuals were seropositive for hMPV by age 2, and almost all individuals 5 years and older were seropositive (59).

Low-pH Triggering of Human Metapneumovirus Fusion: Essential Residues and Importance in Entry

“…RSV F protein activation requires cleavage at two distinct furin sites (25), in contrast to the single furin site present in most F proteins. The HMPV F protein from the majority of strains lacks the ability to be efficiently processed by cellular proteases (47) and must undergo cleavage by extracellular proteases prior to fusion triggering. The absence of HMPV F protein proteolytic processing by intracellular proteases would provide additional control of triggering, preventing the premature activation of F protein in the lower pH environment of the secretory pathway prior to viral assembly.…”

Residues of the Human Metapneumovirus Fusion (F) Protein Critical for Its Strain-Related Fusion Phenotype: Implications for the Virus Replication Cycle

“…Cleavage takes place at a mono-or multibasic cleavage site immediately upstream of a hydrophobic fusion peptide, generating two polypeptide chains (F2 N-terminally located to F1) that remain covalently linked by at least one disulfide bond. It has been reported that the hMPV F protein has a monobasic cleavage site that can be recognized by trypsin (30,32) or by TMPRSS2 (35), a transmembrane serine protease present in the human airway epithelium. The ectodomain of paramyxovirus F proteins also contains two conserved heptad repeat (HR) regions, designated HRA and HRB, which are located downstream of the fusion peptide and upstream of the transmembrane (TM) domain, respectively.…”

The Conserved YAGL Motif in Human Metapneumovirus Is Required for Higher-Order Cellular Assemblies of the Matrix Protein and for Virion Production

“…2C). These results suggested that cell lines that maintain lasting hMPV genome replication could be obtained, especially if the cytopathic effect (CPE) of the virus could be kept minimal, for example, under conditions where the fusion activity of the viral envelope proteins is minimized by avoiding exposure to trypsin (4,70,84). To select cells that harbored replicating hMPV genomes, we fused the GFP marker to the PAC protein that confers resistance to puromycin.…”

“…The presence of the full-length viral genome in these cultures was also demonstrated by the ability to rescue virions, capable of a single cycle of infection, by the ectopic coexpression of wild-type M proteins in the selected cells. Several factors may account for the successful generation of our improved reverse genetics system, including the generation of a single plasmid that carries all four TRC genes instead of the use of four separate expression plasmids and the ability to minimize the CPE of the virus by growing the hMPV-expressing cells in serum-containing medium and in the absence of trypsinization conditions that reduce the fusogenic activity of the F protein (4,70,84). However, it should be noted that even under these conditions, the initial number of surviving colonies was relatively small (about 10 colonies for each transfection).…”