Characterization of a Slow‐Migrating Component of the Rabies Virus Matrix Protein Strongly Associated with the Viral Glycoprotein

Abstract

The envelope of the rabies virus (a prototype of the Rhabdoviridae) contains two species of virus-coded major polypeptides; a transmembrane glycoprotein (G) and a matrix protein (M; formerly called M2), and some host-derived minor components including CD44 and a CD99-related glycoprotein (VAP21) (20,36,37,40). The M protein is a small non-glycosylated internal virion protein (Mr.ϭ24 kDa), and is thought to be located underneath the lipid bilayer of the envelope, where the M protein is thought to associate with the viral G protein and also the viral nucleoprotein (RNP) (41).The G protein has been studied extensively for many years because of its possible roles in determining the host range, neurovirulence, and immunogenicity of the virus as well as important roles in the virus replication cycle

Two Different Conformations of Rabies Virus Glycoprotein Taken under Neutral pH Conditions

“…These results were well consistent with the data obtained in our previous report of a similar two-step IP assay (26), in which we compared the coprecipitated G proteins recovered with anti-M mAb (#3-9-16) and pAb, showing that similar amounts of G protein were coprecipitated with either anti-M mAb or pAb. To be noted specially is that anti-M mAb #3-9-16 recognizes only the β-form of M protein (Mβ), one of the two forms of M protein that exposes the 3-9-16 epitopecontaining N-terminus, and is present mostly in a dimer form in the virion as well as in the cell (26). From these observations and considerations, we assumed that the acid-resistant fraction of the B form of G proteins was intimately associated with the M protein (probably the dimer form).…”

“…A portion of those G proteins, however, seemed to be limited in such changes, probably due to the intimate association of a dimer form of M protein, resulting in preserving its ability to keep the 1-30-44 epitope-positive conformation even when exposed to acidic conditions. These results are consistent with our previous reports describing the strong (i.e., NP-40/DOC-resistant) G-M association, for which a C-terminal-sided small region of M protein is essential (25), and for which a dimer form of the M protein was also suggested to be responsible (26). Possible role(s) of such an intimate G-M association will be discussed.…”

“…The mAbs bound to the cell surface were recovered from the plates by dissolving them in sample lysis buffer for SDS-PAGE, and subjected to 10% SDS-PAGE, followed by electrical blotting to nitrocellulose membrane filters as described previously (26). For estimation of the cell-bound antibodies by semi-quantitative immunoblot assay as described above, the blotted membrane filter was subjected to normal blocking procedures with skim-milk, and then incubated with the first antibody against the murine immunoglobulin, followed by incubation with the horseradish peroxidaseconjugated second antibody and chemiluminescent immunoblotting assay by luminol oxidation.…”

“…3), the 1-30-44 epitopepositive B form did not disappear completely under lower pH conditions, but 15-20% of the G proteins were still detected as the B form in terms of reactivity to mAb #1-30-44 even at pHs as as low as 5.8 to 6.0, at which the fusion-active A form would be predominant. This point was further investigated from the viewpoint of stabilization of B form by its intimate association with the matrix (M) protein, since we observed previously that a portion of G proteins were present in association with the viral M protein (25,26).…”

Genetic Analysis of Phosphoprotein and Matrix Protein of Rabies Viruses Isolated in Brazil

“…The M protein binds to the RNP core, and collaborates with the viral G protein in the infected cells to produce progeny virions by budding at the cell membrane [34,35]. The M protein is also required for the development of a typical bullet-shaped rhabdovirus [29].…”