J. gen. Virol. (1987), 68, 1533-1542. Printed in Great Britain Key words: BDMV/capsid stability/polyamines 1533 Stability of Belladonna Mottle Virus Particles: the Role of Polyamines and Calcium By HANDANAHALSUBBARAO SAVITHRI, 1. SANJEEV KUMAR MUNSHI,-" SANKURATRI SURYANARAYANA, t SOUNDARDIVAKAR 2 AND MATHURR. N. MURTHY 2 1Department of Biochemistry and 2Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India (Accepted II March 1987) SUMMARY The stability of belladonna mottle virus (BDMV) has been studied with respect to elevated pH and to freezing and thawing. BDMV, purified by a modified procedure, was stable at alkaline pH, in contradiction to earlier reports. This difference in the stability could be attributed to the presence of 90 to 140 molecules of spermidine, 20 to 50 molecules of putrescine and 500 to 900 calcium ions in each virus particle. The polyamines could be easily exchanged with other cations such as potassium or caesium and this resulted in a loss of particle stability. These cations may therefore play a role in maintaining the integrity of particle structure. The formation of empty protein shells as a result of freezing and thawing BDMV particles parallels earlier observations on turnip yellow mosaic virus particles. INTRODUCTION Belladonna mottle virus (BDMV, Moline & Fries, 1974) belongs to the tymovirus group of monopartite RNA plant viruses. The nucleic acid in these viruses is encapsidated in a T = 3 icosahedral shell of protein subunits with an approximate Mr of 20 000. The type member of this group, turnip yellow mosaic virus (TYMV) has been extensively studied in terms of the forces that stabilize the particle structure. The virions of TYMV are stabilized by strong hydrophobic interactions between the protein subunits (Kaper, 1971, 1975). Neutron scattering experiments revealed that there is little or no penetration of nucleic acid into the densely packed protein coat of TYMV (Jacrot et al., 1977). A variety of conditions, such as pH above 11-5, freezing and subsequent thawing, elevated temperatures around 45 °C or exposure to certain chemical agents, result in the release of nucleic acid and formation of empty shells from the virions of TYMV (Kaper, 1960, 1971, 1975; Katouzian-Safadi et al., 1980; Katouzian-Safadi & Berthet- Colominas, 1983; Keeling & Matthews, 1982). While TYMV has been the subject of many detailed studies, other members of this group of viruses have been examined in less detail. Recently, it has been shown that RNA is released from BDMV at alkaline pH but that the addition of polyamines and cations prevents this release (Virudachalam et al., 1983a, b). However, BDMV purified in our laboratory by a modified procedure was stable at alkaline pH. This observation prompted a detailed examination of the stability of BDMV particles. The results presented in this paper highlight the role of polyamines and other cations in stabilizing the particle structure and provide an explanation for the instability of BDMV isolated earlier (Virudachalam et al., 1983a). METHODS Isolation ofBDMV. BDMV was propagated in either Nicotiana glutinosa or N. clevelandff or the hybrid N. clevelandii × N. glutinosa (Christie, 1969)plants. Infected leaves were harvested 10 to 14days after inoculation and homogenized in either 0-05M-sodiumcitrate or 0-05 M-sodiumacetate buffer at pH 5.5. The virus was purified by 0000-7572 © 1987 SGM