Enzymic Sensitivity of Conformationally Altered Virions of Southern Bean Mosaic Virus

Abstract

Pancreatic ribonucleasc and sucrose (ribonuclease-free) were purdiased from Schwarz/ Mann (Orangeburg, NJ). Ribonucleases S and N, bacieriophagc MS2 and Esdjeridjia coli iRNA and rRNAs were obta ned from Miles Laboratories (Elkhart, IN). Pronasc was supplied by Calbiodiem (Los Angeles, CA), and proteinase K was obtained from EM Laboratories (Elmsford, NY). All other enzymes were purdiased from Wonhington Biochemical Corporation (Freehold, NJ).SBMV virions (bean strain) were propagated in Phaseolus vulgaris h. 'Bountiful' and purified according to Hsu, SEHGAL and PICKETT (1976). SBMV-RNA was isolated and purified by the procedure of Hsu, WHITE and SEHGAL (1977). Infectivity assays were performed on the primary leaves of P. vulgaris 'Pinto' (SEHGAL 1973a). Virions (2-3 mg/ml) were treated (5°C, 1 h) with EDTA (15 mM) dissolved in 0.1 M sodium phosphate buffer, pH 7.5. All enzyme treatments were done at 5 ^C for I h.The following procedure was used in removing nueleases from the treated virions prior to RNA extraction. To the virion suspension, sucrose (3 %) was added and this sample (2.5 ml) was layered on a 2 ml suerose cushion (10'}?} in a 1.3X6.4cm uhracentrifuge tube. The remainder of the tube was filled with 0.02 M phosphate buffer, pH 7.5. After centrifugation at 27,000 rpm (rotor #30, using 6.5 ml adapter, Bcdtman Instruments, Inc., Palo Alto, CA) for 3 h at 5'^'C, the virion pellets were washed gently with 0.02 M phosphate buffer, pH 7.5, and then suspended (ca. 6-8 mg/ml) in the same buffer. Approximately, 80-95% of the virus was recovered by this procedure judging from A^^^ values. Virions were then exposed to the pH 7.5-dissociative medium (0.1 M phosphate buffer pH 7.5, containing 1 mM EDTA, 1 % SDS, and purified bentonite, 50 //g/ml), to release RNA. These samples were then analyzed with density gradient sedimentation (SEHGAL 1973b, Hsu, WHITE andSEHGAL 1977).Sucrose density gradient centrifugations were performed in linear 5-30% gradients (prepared in 0.02 M phosphate buffer, pH 7.0), E.coli tRNA (5 S), rRNA's (17S and 23 S), bacteriophage MS2 (DOS), and tobacco mosaic virus (190S), served as the sedimentation markers, Polyacrylamide gel (5%) electrophoresis for SBMV protein was performed in the neutral-SDS buffer system of MAIZEL (1969) and molecular weight estimations were made according to WRBER, PRINGLE and OSBORN (1972). Negatively stained samples were examined with a Jeol 100 B electron microscope (SEHGAL and Hsu 1977). Ouditerlony double diffusion tests were performed according to SEHGAL and SINHA (1974).

Sobemoviruses

“…A low-molecular-weight RNA component and coat protein formed Tϭ1 particles at acidic and neutral pHs. It is possible to convert swollen SBMV into Tϭ1 particles by digestion with trypsin, which removes the basic N-terminal segment of the coat protein (89). Erickson and Rossmann (19) also showed that Tϭ1 particle formation did not require RNA when purified and partially digested coat protein, lacking the basic N-terminal portion, was used.…”

Coat Protein‐derived Small Particles in a Tombusvirus from River Lato

“…Small particles similar in size to those associated with LRV, were artificially derived from icosahedral (T = 3) RNA plant viruses by chemically or enzymancally degrading their virions (LEBERMAN and FINCH 1970, TREMAINE and RONALD 1978, SEHGAL et al 1979, ERICKSON and ROSSMAN 1982, SAVITHRI and ERICKSON 1983, TREMAINE et al 1983. With LRV, we were unable to produce artificially small particles although they were constantly found in virus preparations stored at 5 °C over a two-year observations.…”

“…Attempts were made to artificially derive small particles from freshly purified LRV preparations using aikaHne buffers (100 mM Tris-500 mM KCl, pH 9.5; 100 mM Tris-100 mM NaCi, pH 8.2), FDTA (1-5 mM EDTA, pH 8) or enzymatic treatments (2-0.2 % of virus weight of the following: Trypsin, Proteinase K, a-Chimotr^'psin) under the conditions used by several authors (LEBERMAN and FINCH 1970, TREMAJNE and RONALD 1978, SEHGAL et al 1979. All treatments affected to various extent the integrity of LRV particles from moderate swelling to complete destruction ( Fig.…”

Nucleotide sequence of the bean strain of southern bean mosaic virus

“…Factors affecting stabilization of the capsid of SBMV represent a common link with other members of the sobemovirus group and have been extensively studied (Hull, 1977(Hull, , 1978Sehgal et al, 1979;AbdeI-Meguid et al, 1981;Kruse et aL, 1982;Brisco eta/., 1986;Sehgal, 1990). The function of metal ions, in particular of calcium and magnesium, in relation to the assembly and stabilization of SBMV protein shells has been investigated in both the bean and cowpea strains (Hsu et al, 1976;Hull, 1977).…”