Signal Peptide and Helical Bundle Domains of Virulent Canine Distemper Virus Fusion Protein Restrict Fusogenicity

Abstract

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F 1 subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Canine Distemper Virus Infects Canine Keratinocytes and Immune Cells by Using Overlapping and Distinct Regions Located on One Side of the Attachment Protein

“…Although virulent CDV induces only rare syncytia in keratinocytes, we noticed that coexpression of both F wt and H wt glycoproteins in keratinocytes resulted in significant induction of cell-to-cell fusion activity. This could be explained by the fact that (i) in transient-transfection assays, fusion activity occurs in the absence of the matrix protein and/or that (ii) we used an engineered version of F that contains a heterotypic signal peptide; both characteristics have been reported to correlate with enhancement of fusion activity (23,42).…”

“…Western blotting was performed as described previously (23). Transfected cells were washed twice with cold phosphate-buffered saline (PBS) before the addition of 150 l of lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS]) with Complete protease inhibitor (Roche Biochemicals).…”

“…The quantitative fusion assay was performed as described previously (18,23). Briefly, Vero cells were cotransfected with the F and H expression plasmids and 0.1 g of pTM-Luc (kindly provided by Laurent Roux, University of Geneva).…”

Two Domains of the V Protein of Virulent Canine Distemper Virus Selectively Inhibit STAT1 and STAT2 Nuclear Import

“…Western blotting. Immunoblotting was performed as previously described (32). Briefly, Vero-SLAM cells were cultured in six-well plates and infected (with the various recombinant CDVs [rCDVs]) at an MOI of 0.02 1 day later or transfected with the different expression vectors.…”

Region between the Canine Distemper Virus M and F Genes Modulates Virulence by Controlling Fusion Protein Expression

“…Shortening of the Fsp results in increased syncytium formation, indicating that this region regulates the F protein function (30). Further studies have shown that this fusion-enhancing effect is observed only in the context of the Onderstepoort strain vaccine, since shortening of the Fsp originating from the wild-type strain A75/17 did not increase cell-cell fusion (19). Extensive folding and formation of RNA secondary structures due to the high GC content of the CDV M-F region may also contribute to the decrease in F gene expression (15).…”