Robert H. Bussell

Measles, canine distemper and respiratory syncytial virions and nucleocapsids. A comparative study of their structure, polypeptide and nucleic acid composition

Measles, canine distemper (CDV) and respiratory syncytial (RS) virus nucleocapsids were isolated and purified from infected cell cytoplasm, and virions were processed from the extracellular fluid of infected cell monolayers. Ultrastructurally the nucleocapsids possessed diameters of 17–18 nm with no differences noted between the RS nucleocapsids and other nucleocapsids examined. The nucleocapsids exhibited sedimentation values of from 200–300S and possessed buoyant density values of 1.29–1.30 gm/cc in CsCl. Direct chemical measurements and/or 280/260 nm absorption ratios, showed measles, CDV and RS nucleocapsids to contain 5% RNA. RNA, phenol extracted from [32P]-labelled measles, CDV and RS nucleocapsids was found to be RNase A sensitive, while being DNase resistant. Base composition studies of the [32P]-labelled RNA from these three viral nucleocapsids yielded values of 20–22 mole % for cytosine 21–23 mole % for guanine, 26–28mole % for adenine and 29–31 mole % for uridine. Uridine was always found to be the nucleotide in greatest abundance. Analysis by SDS-polyacrylamide gel electrophoresis (PAGE) of measles and CDV nucleocapsid obtained from infected cells dispersed without enzymes revealed them to contain a single polypeptide with molecular weights of 61000 and 54000 daltons, respectively. The polypeptide patterns of [3H]-fucose and [14C]-amino acid labelled measles and CDV virions were examined by SDS-PAGE and each was revealed to possess at least six polypeptides. At least one of the measles and two of the CDV polypeptides contained carbohydrate. These results demonstrate that measles, CDV and RS viruses have a number of properties in common with the better characterized members of the paramyxovirus group (NDV, Sendai and SV5).

Polypeptide composition of measles and canine distemper viruses

Abstract The proteins of measles and canine distemper viruses were studied by SDS-polyacrylamide gel electrophoresis. Six polypeptides were detected in each virus. These had estimated molecular weights of 79,000; 71,000; 61,000; 53,000; 46,000; and 37,000 daltons. A direct comparison of the number and estimated molecular weights of the polypeptides of Sendai virus made in this study and results previously reported by others for Sendai, SV5 and Newcastle disease viruses reveal marked similarities between measles and canine distemper viruses and the three previously well characterized members of the Paramyxovirus group.

Structural Phosphoproteins Associated with Purified Measles Virions and Cytoplasmic Nucleocapsids

Measles virions and cytoplasmic nucleocapsids were labeled with [3H]-amino acids and [32P]-orthophosphate and were purified from infected Vero cells. When analyzed by PAGE, the two capsid-associated polypeptides (VP2 -- 69,000 daltons, VP3 -- 60,000 daltons) were shown to be phosphorylated. Characterization of the phosphorylated polypeptides by acid hydrolysis and high-voltage paper electrophoresis showed that serine was the major phosphorylated amino acid, although lesser amounts of phosphothreonine were also present. The possible role of phosphorylation in the replication cycle of the virus is discussed.

Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells

Nucleocapsids were isolated and purified from cells infected with measles and canine distemper virus (CDV). Electron microscopy of negatively stained nucleocapsids revealed the mean outside diameter was approximately 17-18 nm in each case and was not significantly different from similar measurements obtained with SV5 nucleocapsids and tobacco mosaic virus (TMV). All nucleocapsids also revealed central hollow cores which ranged from 4.3 to 5.2 nm. Direct chemical determinations showed that both CDV and measles virus nucleocapsids contained about 5% RNA. UV absorption spectra of measles and CDV nucleocapsids were typical for nucleoproteins and 280 260 nm ratios gave estimates of 5% RNA in each case. Buoyant density determinations in CsCl with both unlabeled and 32P-labeled nucleocapsids gave figures of 1.29-1.30 g/cc. Sedimentation analysis by analytical and sucrose gradient centrifugation revealed considerable heterogeneity, with nucleocapsids ranging from about 200-300 S. This heterogeneity was shown by electron microscopy to be due to the highly fragmented nature of the nucleocapsids. Analysis of CDV and measles nucleocapsids by SDS-polyacrylamide gel electrophoresis revealed a single major polypeptide when infected cells were harvested in the absence of proteolytic enzymes. These had estimated molecular weight of 54,000 (CDV) and 61,000 (measles). However, nucleocapsids prepared from cells harvested with the aid of trypsin or pronase contained two polypeptides (38,000 and 24,000 for measles; 27,000 and 22,500 for CDV), while similarly prepared SV5 nucleocapsid contained only a single polypeptide (44,500). These differences in polypeptide composition were correlated with differences in morphologic appearance of the nucleocapsids. Nucleocapsids harvested from cells in the absence of proteolytic enzymes had a loosely coiled, flexible appearance, while those harvested from cells treated with trypsin or pronase had a tightly coiled, rigid appearance. RNA was extracted from 32P-labeled measles and CDV nucleocapsids. Treatment with RNase A converted most of the 32P to a TCA-soluble form and DNase had no effect. Base compositions were determined on the 32P-labeled RNA and both viruses were rich in uracil, the molar ratios for the four bases were similar for each virus and were not significantly different from values reported in the literature for the better-characterized members of the Paramyxovirus group (NDV, Sendai and SV5).

Structural Phosphoproteins Associated with Measles Virus Nucleocapsids from Persistently Infected Cells

Measles virus nucleocapsids were labelled with 3H-amino acids and 32P-orthlls (AV+). When analysed by SDS-PAGE, the two major capsid-associated polypeptides (P, mol. wt. 69,000, and NP, mol. wt. 60,000) were shown to be phosphorylated. Subsequent characterization of the phosphorylated polypeptides by acid hydrolysis and high voltage paper electrophoresis showed that serine and threonine were the major phosphorylated amino acid species. The similarities between the peptide phosphorylation patterns obtained in these studies and those reported earlier for the virus phosphoproteins produced in acute infections (Robbins & Bussell, 1979) indicate that major phosphorylative modifications of the capsid proteins are not involvedin measles virus persistence in AV3 cells.

Cell Fusion by Canine Distemper Virus-Infecied Cells and their Plasma Membranes

Cells first gain detectable fusion induction ability 4-6 h after infection with canine distemper virus even though mature virions are not released until about 10 h. Plasma membranes isolated from cells as early as 6 h after infection also have the ability to induce early fusion. While protein synthesis is not required for fusion in the early fusion assay itself, it is required in infected cells for induction of fusion ability. Early cell fusion by infected cells is apparently the result of synthesis of a virus-induced protein that is found in association with the cell plasma membrane.

Disulfide bonds in Sendai virus glycoproteins.

The precursor of fusion protein (Fo) in Sendai virus growth in Vero cells can be cleaved by trypsin to forms F1 and F2, which can be resolved on SDS-polyacrylamide gels. However, if disulfide bonds are preserved during electrophoresis, F1 and F2 remain linked together even after trypsin treatment (F). Sendai virus growth in embryonated chicken eggs does not contain the precursor Fo. However, an F protein was found for Sendai virus grown in eggs when disulfide bonds were preserved during electrophoresis. The hemagglutinin-neuraminidase (HN) glycoproteins also appear to be disulfide-linked to form large complexes which are observed on SDS-polyacrylamide gels of nonreduced samples.The precursor of fusion protein (Fo) in Sendai virus grown in Vero cells can be cleaved by trypsin to forms Fi and F2, which can be resolved on SDS-polyacrylamide gels. However, if disulfide bonds are