Rat Cytomegalovirus: Studies on the Viral Genome and the Proteins of Virions and Nucleocapsids

Abstract

The nucleocapsids (N-capsids) isolated from the nuclei of rat cytomegalovirus (RCMV)-infected rat embryo fibroblasts (REF) are composed of three major proteins: 142 × 10³ (142K), 40K and 32K mol. wt. Nucleocapsids isolated from the cytoplasmic fraction (C-capsids) are composed of proteins found in N-capsids and five major and seven minor new protein species. Most of the proteins present in C-capsids are found in the extracellular enveloped virions, although the ratios vary. Proteins that are abundantly present, particularly in virions (mol. wt. 125K, 116K, 87K, 79K, 71K, 68K, 62K, 50K, 43K and 28K), are probably the major constituents of the viral envelope. The DNA recovered from extracellular virions was purified to homogeneity and by equilibrium centrifugation in CsCl one density class of 1.716(±0.001) g/ml was found. Contour length measurements showed one size class of a linear double-stranded DNA corresponding to an average mol. wt. of 144(±9) × 10⁶ which is in good agreement with data obtained by restriction endonuclease analysis (REA), which yielded mol. wt. values of 132(±9) × 10⁶ (HindIII), 138(±2) × 10⁶ (EcoRI) and 137 × 10⁶ (BglII). The REA patterns also revealed the presence of 0.25 m and 0.5 m fragments, which might indicate, in analogy with other cytomegalo- and herpesviruses, the existence of four different configurations of the RCMV genome. The infectivity of RCMV DNA was determined in subconfluent REF monolayers. A cytopathic effect characteristic of RCMV was observed 6 days post-transfection and up to 60 plaques/µg DNA were obtained. Using DNA—DNA filter hybridization the degree of homology between the genomes of RCMV and murine or human CMV was examined. Under stringent conditions (50% formamide) values of 12(±2)% and 3(±1)% were found whereas under non-stringent conditions (20% formamide) values of 21(±2)% and 6(±1)% were obtained, respectively.