Measles Virus Envelope Glycoproteins Hetero-oligomerize in the Endoplasmic Reticulum

Abstract

The endoplasmic reticulum (ER) was investigated as the initial oligomerization site for the envelope glycoproteins H and F of measles virus (MV), a clinically relevant member of the Paramyxoviridae family, and consequences of this interaction for viral replication were studied. Both proteins were tagged at their cytosolic tails with RRR and KKXX motifs, respectively, resulting in their efficient retention in the ER. Cotransfection of the retained constructs with transport competent MV glycoproteins revealed a dominant negative effect on their biological activity indicating intracellular complex formation and thus retention. Pulse-chase analysis and co-immunoprecipitation experiments demonstrated that this effect is based on both homo-and hetero-oligomerization in the ER. Recombinant viruses additionally expressing ERretained F showed an altered cytopathic phenotype accompanied by greatly reduced particle release. Similar mutant viruses additionally expressing ERretained H could not be rescued indicating an even greater negative effect of this protein on virus viability. Our study suggests that both homo-and heterooligomerization of MV glycoproteins occur in the ER and that these events are of significance for early steps of particle assembly.

Probing the Spatial Organization of Measles Virus Fusion Complexes

“…Cells transfected with equal amounts of H-Edmand F-Edm-expressing plasmids were treated with the membrane-impermeable, reducible cross-linker DTSSP, followed by quenching and lysis with stringent radioimmunoprecipitation assay buffer. For controls, DTSSP was omitted, cells were transfected with F-Edm-expressing plasmids only or cells were cotransfected with F-Edm and an H-Edm variant harboring a cytosolic endoplasmic reticulum retention motif (H-ER) (55). For immunoprecipitation (IP), a monoclonal antibody cocktail directed against epitopes in the MeV H ectodomain (␣H) was used, followed by brane-distal N-glycans based on our previous identification of the role of MeV H section 110 to 114 in mediating F specificity (36).…”

A target site for template-based design of measles virus entry inhibitors

“…MV stocks were grown and titered as described (29). Simian virus 5 (SV5) and hPIV2 were propagated and titered on Vero, and vaccinia virus was propagated and titered on HeLa cells.…”

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

“…The morbillivirus H protein therefore has a functional motif at its amino-terminus that directs preferential incorporation into viral particles. While morbillivirus F and H proteins associate very early during biosynthesis (Plemper et al, 2001), F also forms VLPs when co-expressed with M (Cathomen et al, 1998a;Pohl et al, 2007), and recombinant MeVs with partially deleted cytoplasmic tails were viable (Cathomen et al, 1998b), it remains unclear which glycoprotein interacts more strongly with the homologous M protein to drive morbillivirus particle formation. In contrast, henipavirus glycoproteins are transported independently and only associate transiently on the cell surface (Lamp et al, 2013;Whitman & Dutch, 2007;Whitman et al, 2009).…”

“…While the morbillivirus F and H proteins associate early during biosynthesis in the ER and are transported to the cell surface as a complex (Plemper et al, 2001), the henipavirus F and G proteins are transported independently (Whitman et al, 2009), indicating that differences in interactions between glycoproteins may influence the assembly and cell-cell fusion process. Paramyxovirus HN/H/G and F protein cytoplasmic domains also contain tyrosine motifs, which result in transport of proteins to the basolateral surface of polarized cells (Runkler et al, 2009;Weise et al, 2010).…”