The Structural Proteins of Rabies Virus and Evidence for their Synthesis from Separate Monocistronic RNA Species

Abstract

Purified preparations of the CVS strain of rabies virus, which were labelled during the infectious growth cycle with different isotopes or labelled in vitro by iodination or acetylation, contained five major proteins, L, G, N, M₁ and M₂, when examined by polyacrylamide gel electrophoresis (PAGE). The major surface glycoprotein, G, could be separated into two components, G₁ and G₂, in some PAGE systems; they were present in about equimolar amounts and had apparent mol. wt. of 70.5 × 10³ and 65 × 10³, respectively. The virus nucleocapsid (ρ = 1.28 g/ml) could be isolated after detergent treatment of purified virus. It contained the virus RNA, the major nucleocapsid protein, N (mol. wt. 58.5 × 10³), and small amounts of a large protein, L (mol. wt. 170 × 10³). Two membrane proteins, M₁ (mol. wt. 39.5 × 10³) and M₂ (mol. wt. 25 × 10³), were also observed. Chromatography of dissociated rabies virus in agarose columns with guanidine hydrochloride did not resolve any additional virus structural proteins. Two-dimensional peptide map analysis of iodinated structural proteins indicated that they were unique gene products and not derived from a precursor polypeptide by cleavage. The peptide maps of the two glycoproteins, G₁ and G₂, appeared identical. The approximate number of protein molecules per virion has been determined. Rabies virus-directed protein synthesis in BHK21 cultures was detected as early as 6 h p.i. and all the proteins were present 12 h p.i. Additional non-structural virus-specific proteins were not observed. The NaCl hypertonic shock procedure, which differentially inhibits polypeptide chain initiation in different classes of mRNAs, was used to inhibit the synthesis of the G and M₁ proteins relative to the others selectively. All the rabies virus proteins were synthesized simultaneously following release from hypertonic treatment, suggesting that there are independent polypeptide chain initiation sites for the synthesis of each of the rabies proteins and that each protein is derived via translation of monocistronic mRNA species.