Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner

Abstract

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Looking at the Pathogenesis of the Rabies Lyssavirus Strain Pasteur Vaccins through a Prism of the Disorder-Based Bioinformatics

“…Being located on the surface of RABV particles, the G-protein controls receptor binding and the release of the viral ribonucleoprotein (RNP) in the cytoplasm via pH-dependent membrane fusion, thereby playing a crucial role in the cell entry and in vivo spread [ 128 ]. Furthermore, it was shown that the G-protein (in particular, its ectodomain) accumulates adaptive mutations that improve the release of infectious viral particles [ 129 ].…”

Comparison of Pan-Lyssavirus RT-PCRs and Development of an Improved Protocol for Surveillance of Non-RABV Lyssaviruses

“…Of note, we employed RNA transcripts as standard for multiple reasons, among them the lower biosafety level required and the reliability of the target sequence compared to cell-adapted strains. Indeed, lyssaviruses amplification not only requires biosafety level 3 laboratory facilities, but more importantly induces the fixation of genetic mutations occurring during virus adaptation to a cell substrate, which might critically affect the evaluation of the assay performance [ 39 , 40 ]. Consistently with this, in the framework of previous research activities, in the cell-adapted LLEBV, we observed a G11A mutation within the 3′ non-coding region where LN34/(n)LN34 forward primers bind ( Figure S2 ).…”

Looking at the Pathogenesis of the Rabies Lyssavirus Strain Pasteur Vaccins through a Prism of the Disorder-Based Bioinformatics

“…Being located on the surface of RABV particles, the G-protein controls the receptor binding and the release of the viral ribonucleoprotein (RNP) in the cytoplasm via the pH-dependent membrane fusion, thereby playing a crucial role in the cell entry and in vivo spread [124]. Furthermore, it was shown that the G-protein (in particular, it's ectodomain) accumulates the adaptive mutations improving the release of infectious viral particles [125].…”