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

Abstract

YXXL motifs in cellular and viral proteins have a variety of functions. The matrix (M) protein of the respiratory pathogen human metapneumovirus (hMPV) contains two such conserved motifs-YSKL and YAGL.We mutated these sequences to analyze their contributions to hMPV infectivity. The mutant clones were capable of intracellular replication; however, the YAGL but not YSKL mutants were defective at spreading in infected cultures. We improved the reverse genetics system for hMPV and generated cell lines that stably expressed selectable, replicating full-length genomes for both the wild type and the mutant clones, allowing microscopic and biochemical analyses of these viruses. YAGL mutants produced normal cellular levels of M protein but failed to release virions, while ectopic coexpression of wild-type M generated particles that were restricted to a single cycle of infection. The YAGL motif did not act as a late (L) domain, however, since hMPV budding was independent of the cellular endosomal sorting complex required for transport (ESCRT) machinery and because replacement of the YAGL motif with classical L domains generated defective viruses. Instead, the YAGL mutants had defective M assemblies lacking a normal filamentous appearance and showed poor extractability from the cell compared to the wild-type protein. The mutant proteins were not grossly misfolded, however, as they interacted with cellular membranes and coassembled with wild-type M proteins. Thus, the YAGL motif is an important determinant of hMPV assembly. Furthermore, the selectable hMPV genomes described here should extend the use of reverse genetics systems in the analysis of spreading-defective viruses.Human metapneumovirus (hMPV) is a respiratory pathogen which causes upper and lower respiratory tract infections worldwide in both healthy and immunocompromised individuals across all age groups, resulting in mild to severe respiratory diseases (reviewed in references 18, 35, and 50). It was first identified in 2001 (84) and is classified, together with avian metapneumovirus (aMPV), as a member of the Metapneumovirus genus within the Pneumovirinae subfamily of the Paramyxoviridae family. The nonsegmented, negative-strand RNA genomes of the MPVs are approximately 13.3 kb long and contain eight genes that are separated by gene start (GS) and gene end (GE) sequences and encode nine putative proteins: nucleoprotein (N), phosphoprotein (P), matrix (M) protein, fusion (F) protein, small hydrophobic surface protein (SH), major attachment glycoprotein (G), major polymerase subunit (L), transcription elongation factor (M2.1), and RNA synthesis regulatory factor (M2.2). The last two proteins (collectively named M2 here) are thought to be expressed from overlapping open reading frames in the M2 gene (16, 83). The overall order of the genes in the hMPV genome is 3Ј-N-P-M-F-M2-SH-G-L-5Ј. hMPV is closely related to the important respiratory pathogen human respiratory syncytial virus (hRSV), which is also classified in the Pneumovirinae subfamily, but the order of the...

Viral Membrane Scission

“…Second, there may be other unknown ESCRT components, pathways, or viral protein substitutes that can function redundantly in certain circumstances and, thus, may compensate when the canonical late-domain sequences are disrupted or the activity of Vps4 is inhibited. This may explain the requirement for late-domain sequences in the budding of a collection of viruses that are insensitive to Vps4 inhibition, including Ebola virus, metapneumovirus (MPV), Newcastle disease virus, Sendai virus, and vesicular stomatitis virus (VSV) (Gosselin-Grenet et al 2007, Irie et al 2004, Neumann et al 2005, Sabo et al 2011, Schmitt et al 2005, Shnyrova et al 2007). Finally, these viruses may use another method of membrane scission in addition to the host ESCRT system, providing a measure of redundancy in the event that one of the scission pathways malfunctions (Figure 4).…”

“…However, a capacity to alter membrane curvature has not yet been demonstrated for Rab11-FIP2, and thus the mechanism of action remains unknown. Interestingly, budding of the closely related avian and human MPV is also ESCRT independent (Sabo et al 2011, Weng et al 2011), though no dependence on Rab11-FIP2 has yet been found for MPV.…”

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

“…The HA-tagged proteins were detected using a primary monoclonal antibody directed against HA (Roche) and a secondary goat anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC). HMPV M protein was detected using an antibody against the avian metapneumovirus C M protein, kindly provided by Sagar M. Goyal (University of Minnesota, Minneapolis, MN), which has been shown to cross-react with HMPV M (40), and a secondary goat anti-rabbit antibody conjugated with TRITC. Pictures were taken using the Nikon 1A confocal microscope and analyzed with the NIS-Elements software.…”

Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

“…MPV encodes 9 proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), M2 with two open reading frames (M2-1, M2-2), small hydrophobic (SH), glycoprotein (G), and polymerase (L). Most of the HMPV proteins are analogous to other paramyxovirus proteins, with analogous functions (Cai et al, 2015; de Graaf et al, 2008; Derdowski et al, 2008; Kitagawa et al, 2010; Leyrat et al, 2014a; Leyrat et al, 2013; Leyrat et al, 2014b; Miller et al, 2007; Sabo et al, 2011; van den Hoogen et al, 2002). …”