Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, “outrunning” the host’s immune response in demyelinating plaques, thus continuously eliciting new lesions

EFFECT OF DETERGENTS ON THE STRUCTURE OF INTEGRAL MEMBRANE-PROTEINS OF SENDAI VIRUS STUDIED WITH SIZE-EXCLUSION HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND MONOCLONAL-ANTIBODIES

The integral membrane proteins of Sendai virus, the fusion protein F (Mr = 65 000) and the haemagglutinin-neuraminidase protein HN (Mr = 68 000), were used as a model protein mixture. They were subjected to size-exclusion high-performance liquid chromatography on Superose 6HR columns with eluents containing various additives in order to solubilize the proteins. The effect of the additives on the structure of the membrane proteins was investigated with conformation-dependent monoclonal antibodies, either directed against F or HN protein, and by determination of the haemagglutinating capacity of the HN protein. The results show that the structure of the HN protein is more easily disturbed by eluents than that of the F protein. When the elution conditions are mild, e.g., 0.1% octylglucoside, the structure of both proteins is conserved but no separation is obtained. Elution with a buffer containing 0.05% sarkosyl (dodecyl methylglycine sodium salt) did not affect the structure and resulted in pure F protein. Pretreatment of the Amberlite XAD-2-treated Sendai virus envelope extract with 4% sodium dodecyl sulphate (SDS) and elution with 0.1% SDS in 50 mM sodium phosphate (pH 6.5) altered the structure of the HN protein but resulted in purification of the tetramer and the dimer of the HN protein, and the monomer of the F protein