Analysis of structural polypeptides of purified human cytomegalovirus

Abstract

Human cytomegalovirus strain C87 was purified. Extracellular virus was concentrated by centrifugation at 100,000 .times. g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. After 2 consecutive isopycnic potassium tartrate gradient centrifugations (20-50%), coinciding peaks of plaque titer, protein and radioactivity were found at a density of from 1.20-1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from 2 mixtures: [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e., about 2.5% of the protein in the purified virus preparation could be accounted for by host [human fetal tonsil cells] protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VP) and their MW ranged from 11,000-290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine-labeled virus showed six bands. Four of these were so broad that several VP corresponded to each of the glycosylated bands. When heavy (2 fractions close to 1.21 g/cm3) and light (2 fractions close to 1.20 g/cm3) fractions of the Pfu peak from the 2nd potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.