K. W. Buck

Biophysical Properties of Penicillium stoloniferum Virus S.

Summary Pencillium stoloniferum virus S has been fractionated into four particle classes, E, M, L and H, with sedimentation coefficients of 66, 87, 101 and 113 S, respectively. E particles were shown to be empty capsids, while M particles contained single-stranded RNA, L particles contained double-stranded RNA, and H particles contained both single-stranded RNA and double-stranded RNA. The mol. wt. of L particles was found from sedimentation and diffusion coefficients to be 6.0 × 106. Evidence is presented that the M, L, and H particle classes each contain two components, M 1, M 2, L 1, L 2, H 1 and H 2, respectively. It has been shown that the M 1, M 2, L 1 and L 2 particles each contain only one molecule of RNA with mol. wts. of 0.47 × 106, 0.56 × 106, 0.94 × 106 and 1.11 × 106, respectively, while H 1 and H 2 particles each contain one molecule of double-stranded RNA of mol. wts. 0.94 × 106 and 1.11 × 106, respectively, together with single-stranded RNA.

Priming of Complementary DNA Synthesis in Vitro by Small DNA Molecules Tightly Bound to Virion DNA of Wheat Dwarf Virus

Summary DNA isolated from purified preparations of wheat dwarf virus (WDV) has been shown to contain tightly bound small DNA molecules which can act as primers for the synthesis of full-length complementary DNA in vitro. The small DNA molecules are bound in the terminating intergenic region of the WDV genome between the end of an open reading frame encoding a putative protein of M r 17292 and a conserved ‘A-T’ box containing putative transcriptional polyadenylation signals. Evidence that the small DNA molecules contain ribonucleotides at their 5′ termini is presented and their possible role in the priming of virus DNA synthesis in vivo is discussed.

Agroinfection of Triticum aestivum with Cloned DNA of Wheat Dwarf Virus

Summary A head-to-tail dimer of cloned DNA from a Swedish isolate of wheat dwarf virus (WDV) was integrated between the T-DNA border sequences of a broad host range binary vector and transferred into cells of wheat seedlings using an Agrobacterium-mediated delivery system. Two-thirds of the inoculated plants developed a systemic infection. Extracts of infected leaves were shown to contain the virus double-stranded (supercoiled, open circular and linear) and single-stranded DNA forms of unit genome length and the virus capsid polypeptide. The results demonstrate the infectivity of a previously sequenced clone of WDV DNA.

Biophysical and biochemical properties of two viruses isolated from Aspergillus foetidus.

Aspergillus foetidus virus S (AfV-S) and A. foetidus virus F(AfV-F) have been shown to be serologically unrelated. Amino acid compositions of the two virus capsids are compared and their capsid polypeptides have been examined by SDS-polyacrylamide gel electrophoresis. AfV-F contained one major (phi3) and two minor (phi1 and phi2) polypeptides with mol. wt. 87000, 125000 and 100000, while AfV-S contained one major (sigma1) and one minor (sigma2) polypeptide with mol. wt. 83000 and 78000 respectively. Evidence is presented that sigma2 may be derived from sigma1 polypeptide by proteolytic degradation in vitro. The mol. wt. of AfV-F4 and AfV-S1a particles were found from sedimentation and diffusion coefficients to be 13 I times 10-6 and 12 4 times 10-6 respectively. AfV-F capsid was estimated to contain 120 molecules of polypeptide phi3 and one molecule each of polypeptides phi1 and phi2, while AfV-S capsid was estimated to contain 120 molecules of polypeptide phi1. It has been shown that SIa and S-2a particles each contain a molecule of double-stranded RNA with mol. wt. 2 24 times 10-6 (RNA-224) and 2 76 times 10-6 (RNA-276) respectively, whereas S-Ib and S-2b particles each contain a molecule of RNA-224 and RNA-276 respectively, together with an additional molecule of double-stranded RNA of mol. wt. 0 I times 10-6. Evidence is presented that S4 particles contain two molecules of RNA-224. S3 particles gave only RNA-224 on extraction, but contain the equivalent of 11/2 molecules of RNA-224; the nature of these particles and other possible virus replicative intermediates is discussed. Double-stranded RNA of mol. wt. I 24 times 10-6 was derived from a newly described particle class, Fo.

Virus Particles in Aspergillus foetidus: a Multicomponent System

Summary Two electrophoretically distinct classes of virus particles, designated fast (AfV-F) and slow (AfV-S) according to their relative electrophoretic mobilities, were isolated from the mycelium of Aspergillus foetidus, strain imi 41871. They were almost completely separated by dialysis against 0.03m-phosphate buffer, pH 7.6, in which F particles remained in suspension, while S particles precipitated, and were further purified by caesium chloride density gradient centrifugation. Electron microscopy showed that both classes were composed of isometric particles of similar diameter. RNA, prepared from both AfV-F and AfV-S, was double-stranded. In polyacrylamide gel electrophoresis AfV-F RNA was resolved into four main components with molecular weights of 2.31, 1.87, 1.70 and 1.44 × 106, while AfV-S RNA gave two components with molecular weights of 2.76 and 2.24 × 106. Both AfV-F and AfV-S could be separated by centrifugation in caesium chloride gradients into a number of fractions containing particles with different buoyant densities. Electrophoretic analysis of the RNA prepared from the virus fractions indicated that the multiple RNA components are not fragments released from a single virus particle, but are separately encapsidated in different particles.

Comparison of interferon induction in mice by purified Penicillium chrysogenum virus and derived double-stranded RNA.

Summary A virus preparation, obtained from the mycelium of Penicillium chrysogenum, strain Q176, was purified by isopycnic centrifugation on linear gradients of potassium tartrate. Double-stranded RNA, prepared from the virus particles, revealed three components on electrophoresis on polyacrylamide gels. The serum interferon levels in mice after intravenous injection of free virus RNA were higher and earlier than for purified virus particles, containing an equivalent amount of RNA. After reaching their maxima, the interferon activities declined at similar rates to very low levels after 45 hr for free RNA and 39 hr for virus particles.

Serological Studies on Tomato Golden Mosaic Virus, a Geminivirus

Summary Particles of tomato golden mosaic virus (TGMV), purified by an improved procedure, were used to prepare an antiserum which in gel double-diffusion tests with homologous virus gave a single precipitin line and had a titre of 1/256. TGMV was shown to be serologically related to another geminivirus, cassava latent virus, and distantly related to a leafhopper-transmitted geminivirus, beet curly top virus. No relationship could be detected to four other leafhopper-transmitted geminiviruses.

Replication of Red Clover Necrotic Mosaic Virus RNA in Cowpea Protoplasts: RNA 1 Replicates Independently of RNA 2

Summary Inoculation of cowpea mesophyll protoplasts with unseparated RNA 1 and RNA 2 from red clover necrotic mosaic virus in the presence of polyethylene glycol resulted in virus RNA replication, the synthesis of virus capsid polypeptide and the formation of virus particles; 75 to 85% of the viable protoplasts became infected and the yield of virus particles or virus RNA after 72 h incubation corresponded to about 3 × 106 virus genomes per infected protoplast. In contrast, no replication could be detected when protoplasts were inoculated with RNA 2 alone. However, inoculation of protoplasts with RNA 1 alone resulted in its replication and the formation of virus particles, with a yield similar to that obtained after inoculation with both RNAs. Since infection of plants requires both RNA 1 and RNA 2 to be present, the demonstration of the independent replication of RNA 1 in single cells strengthens the hypothesis that RNA 2 plays a role in the cell-to-cell transmission of the virus.

Comparison of the Biophysical and Biochemical Properties of Penicillium cyaneo-fulvum Virus and Penicillium chrysogenum Virus

The biophysical and biochemical properties of Penicillium cyaneo-fulvum virus (Pc-fV) and Penicillium chrysogenum virus (PcV) have been compared. In sucrose density gradient sedimentation, purified virus preparations gave one major component, L, and three minor components E1, E2 and H with sedimentation coefficients of 145S, 80S, 102S and 172S respectively in each case. E1, E2 were shown to be empty particles. Pc-fV L particles contained only double-stranded RNA, which separated in polyacrylamide gel electrophoresis into four components with mol. wt. 2-21 X 10(6), 2-08 X 10(6), 1-98 X 10(6) and 1-93 X 10(6). PcV L particles gave three double-stranded RNA components, which were indistinguishable in polyacrylamide gel electrophoresis from the larger three RNA components of Pc-fV. In both viruses each RNA component was separately encapsidated. In both viruses H particles gave rise to the same double-stranded RNA components as their L particles, but contained, in addition, small amounts of single-stranded RNA. Pc-fV and PcV have been shown to consist of isometric particles of the same size and to be serologically related, and the amino acid compositions of their capsid polypeptides were found to be very similar. The capsid polypeptides of the two viruses were examined by SDS-polyacrylamide gel electrophoresis. In each case E2, L and H particles gave one major polypeptide gamma1, with mol. wt. 130000, whereas E1 particles contained one major polypeptide gamma2 with mol. wt. 115000. The mol. wt. of L particles, determined from sedimentation and diffusion coefficients, was 9-8 X 10(6) for both Pc-fV and PcV. The capsid of the L particles of each virus was estimated to contain 60 molecules of polypeptide gamma1.

RNA 2 of Red Clover Necrotic Mosaic Virus Determines Lesion Morphology and Systemic Invasion in Cowpea

Summary Red clover necrotic mosaic virus (RCNMV) has two RNA species of mol. wt. about 1.5 × 106 (RNA 1) and 0.5 × 106 (RNA 2). An English strain (H) and a Czechoslovakian strain (TpM-34) of RCNMV could be distinguished serologically, by solid-phase RNA hybridization analysis (Northern blotting) and by the symptoms they induced in cowpea. Studies of pseudorecombinants, formed following inoculation of plants with heterologous combinations of the RNA species of each strain, showed that RNA 2 determines the morphology of lesions induced by the isolates in cowpea and their ability to invade the plants systemically.