Buoyant density and morphology of rubella hemagglutinin.

Rubella virions demonstrated by negative-contrast electron microscopy have recently been reported to range from 500 to 750 A in diameter and to have amorphous pleomorphic envelopes (J. M. Best et al., Lancet 2:237, 1967). Consonant with these observations, sedimentation studies have indicated a diameter of 850 A and ultrafiltration experiments, 900 A (B. Russell et al., J. Gen. Virol. 1:305, 1967). In equilibrium centrifugation in CsCl gradients with an initial density of 1.23 g/cm3, the infectious moiety has been reported to have a buoyant density of 1.23 g/cm3 (N. J. Schmidt et al., J. Immunol. 99:399, 1967). In gel filtration experiments, infectious virus has been eluted from Sephadex G-200 together with the large complement-fixing (CF) antigen shortly after displacement of the column void volume (N. J. Schmidt et al., Proc. Soc. Exptl. Biol. Med. 123:758, 1966). In the present study, density gradient centrifugation in CsCl, gel filtration in Sephadex G-200, and electron microscopy were employed in attempts to characterize the rubella virus hemagglutinin. Rubella hemagglutinating antigen (HA) was prepared from strain RA/27/3 (13) rubella virus grown in suspension cultures of BHK-21 cells maintained in an inhibitor-free medium [P. E. Halonen et al., Ann. Med. Exptl. Biol. Fenniae (Helsinki) 45:182, 1967]. Infected cultures were harvested after 5 days of incubation and were clarified by centrifugation at 1,800 rev/min for 10 min. HA antigen was concentrated from the resulting supernatant fluid by centrifugation at 30,000 rev/min for 4 hr in a no. 30 Spinco rotor. The pelleted antigen was resuspended in distilled water to 4% of the original volume. HA tests were performed at 4 C with the use of 0.25% chick erythrocytes at a final pH of 6.0, according to a standardized procedure adapted to the microtiter system [P. E. Halonen et al., Ann. Med. Exptl. Biol. Fenniae (Helsinki) 45:182, 1967]. For CsCl density gradient centrifugation, concentrated HA antigen was mixed with a stock solution of CsCl of density 1.80 g/cm3 to yield starting densities of approximately 1.25 g/cm3. The HA antigen-CsCl mixtures were subjected to centrifugation for 36 hr at 39,000 rev/min in a SW-39 rotor. Fractions were collected from the bottom of the tubes with a fraction collector, and fraction densities were determined immediately by weighing 100 uliter samples. Results of equilibrium centrifugation of the HA antigen are presented in Fig. 1. As shown, a sharp peak of HA activity was recovered in fraction no. 3 which had a