R.I.B. Francki

Towards a system for the identification and classification of potyviruses I. Serology and amino acid composition of six distinct viruses.

Abstract Antigenic relationships of six distinct potyviruses were studied by immunodiffusion tests using highly purified sonicated virus preparations and anti-intact virus sera devoid of detectable antibodies to host-plant antigens. Three variants of bean yellow mosaic virus (BYMV) including BYMV sensu stricto and two variants of pea mosaic virus (PMV and SPMV) were shown to be antigenically very similar and also relatively closely related to lettuce mosaic virus (LMV). Distant antigenic relationships were detected between the BYMV variants and bean common mosaic virus (BCMV); between BCMV and passionfruit woodiness virus (PWV); and between PWV and potato virus Y (PVY). No antigenic relationships were detected between any of these viruses and sugarcane mosaic virus (SCMV). Antibodies in anti-viral sera were very poor in recognizing coat proteins dissociated with LiCl from homologous viruses and failed altogether to recognise those dissociated with pyrrolidine. Attempts to prepare antisera in mice against isolated viral coat proteins dissociated with either LiCl or pyrrolidine were unsuccessful due to poor immunogenicity of the preparations. Electrophoretic mobilities of the viral coat proteins relative to marker proteins in the presence of sodium dodecyl sulphate suggest that the protein subunits of all the viruses studied have molecular weights of about 33,000. However, the coat proteins were prone to partial degradation. The amino acid compositions of the antigenically closely related viruses were very similar, but similarities of those distantly related were no greater than those of the apparently unrelated viruses. The problems in the use of serological and amino acid composition data obtainable with currently available techniques for the classification of potyviruses are discussed.

Comparative studies on tomato aspermy and cucumber mosaic viruses: I. Physical and chemical properties

Abstract Some physical and chemical properties of the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) have been compared. The size, morphology, sedimentation rate, RNA base ratio, and buoyant density of the two viruses are indistinguishable. Preparations of RNA from both viruses were each resolved into four distinct species by polyacrylamide-gel electrophoresis. TAV-RNA preparations contained species with molecular weights of 1.26 × 10 6 ,1.10 × 10 6 , 0.90 × 10 6 , and 0.43 × 10 6 daltons, and CMV-RNA, species of 1.26 × 10 6 , 1.10 × 10 6 , 0.77 × 10 6 , and 0.34 × 10 6 daltons. Analysis of sodium dodecyl sulphate (SDS)-treated viral proteins by polyacrylamide-gel electrophoresis showed that both viruses have protein subunits of molecular weight 24,500 daltons. The amino acid compositions of proteins from the two viruses, although similar, were distinguishable, and the calculated molecular weights of protein subunits were 26,100 and 26,300 daltons for TAV and CMV, respectively. The two viruses were serologically distinct. On the data presented it is suggested that in preparations of both TAV and CMV three distinct particles are present in each with identical protein shells, but different RNA cores.

Studies on encapsidated viroid-like RNA I. Characterization of velvet tobacco mottle virus

Velvet tobacco mottle virus (VTMoV) isolated from Nicotiana velutina growing wild in arid Central Australia was transmitted by inoculation to a limited number of plant species of which N. clevelandii was the most convenient experimental host. The virus was also transmitted from field-grown plants toN. velutina and N. clevelandii by the mirid Cyropeltis nicotianae. VTMoV preparations purified by clarification with organic solvents and differential centrifugation contained polyhedral particles about 30 nm in diameter sedimenting as a single component at about 115 S. The particles were shown to be located in the nucleus, cytoplasm, and vacuoles of infected plant cells. Virus dissociated in the presence of mercaptoethanol and sodium dodecyl sulfate (SDS) separated into one major and two minor polypeptides with estimated molecular weights of 33,000, 36,000 and 31,000, respectively. Single-stranded RNA isolated from VTMoV by extraction with phenol was separated into five components with apparent molecular weights of 1.5 x 10(6), 0.63 x 10(6), 0.25 x 10(6), 0.16 x 10(6), and 0.12 x 10(6) referred to as RNAs 1, 1a, 1b, 2, and 3, respectively. It appears that RNAs 1a and 1b are breakdown products of RNA 1, as shown elsewhere, and electron microscopic examination of the other species showed that whereas RNAs 1 and 3 are linear molecules, RNA 2 is circular. The similarity of RNAs 2 and 3 to the RNA of viroids is discussed. VTMoV has been compared with several RNA plant viruses with small polyhedral particles. Only solanum nodiflorum mottle virus appears to share some of its unique features and the two have been shown to be antigenically related.

Comparative studies on tomato aspermy and cucumber mosaic viruses: III. Further studies on relationship and construction of a virus from parts of the two viral genomes

Although the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) do not show any detectable serological relationship or nucleotide base sequence homology, it was possible to construct a pseudo-recombinant virus from parts of their RNA genomes. The pseudo-recombinant from the two largest RNA molecules of TAV (T1 and T2) when combined with the third largest molecule of CMV (C3) formed a stable virus whose coat protein was like that of CMV, but it induced symptoms on several plants indistinguishable from those produced by TAV. It is concluded that TAV and CMV have functionally divided genomes and that the coat protein cistron is located on RNA component 3, whereas the cistron or cistrons determining host reactions are located on either RNA component 1 or 2, or on both 1 and 2.

Some properties of purified cucumber mosaic virus (Q strain).

Abstract Strain Q of cucumber mosiac virus (QCMV) from Queensland, Australia, contains 18% ribonucleic acid (RNA) which has a base ratio (moles %) of adenine 22.4%, guanine 24.7%, cytosine 22.8%, and uracil 30.1%. The molecular weight of the virus is approximately 5.8 × 10 6 , based on a sedimentation coefficient of 98.6 S and a diffusion coefficient of 1.20 × 10 7 cm 2 per second. Hence the virus contains about 1.0 × 10 6 molecular weight units of RNA. The electrophoretic mobility of QCMV is −7.2 × 10 −5 cm 2 per volt per second at pH 9 and −4.0 × 10 −5 cm 2 per volt per second at pH 7.4. Electron micrographs of QCMV preparations show particles about 28 mμ in diameter when stained in phosphotungstic acid (PTA) or uranyl acetate and about 42 mμ when shadowed with gold-palladium. Partially purified QCMV in 0.005 M borate buffer, pH 9, remained infectious for several months when stored at 0–4°C. Such preparations retained some infectivity after heating between 70 and 80° for 10 minutes whereas freshly extracted sap from infected cucumber plants was completely inactivated after 10 minutes at 60°. QCMV precipitated in the pH range 4.3–5.5. At 0°C, QCMV was unaffected by the presence of NaCl at concentrations up to about 0.25 M , but was rapidly precipitated on mild heating. QCMV is unstable and slowly disrupts in 2% PTA, pH 7. It is also degraded in 1 M CaCl 2 and 2 M LiCl and the RNA precipitates quantitatively leaving most of the protein in solution. CaCl 2 -precipitated QCMV-RNA could not be dissolved in neutral buffers, but the RNA precipitated with LiCl was readily resuspended and shown to be several times more infectious than phenol-extracted RNA. QCMV has been shown to be antigenically similar to the Y strain of CMV (CMV-Y) from North America. However, these two strains appear to differ in some respects which are discussed.

Comparative studies on two satellite RNAs of cucumber mosaic virus.

Cucumber mosaic virus (CMV) satellite RNA (Sat-RNA, D. W. Mossop and R. I. B. Francki, 1978, Virology86, 562-566) is similar in many of its physical and biological properties to CMV associated RNA 5 (CARNA 5) described by Kaper and Tousignant (1977, Virology85, 323-327). However, CARNA 5, unlike Sat-RNA, causes a serious necrotic disease of tomato. Sat-RNA when inoculated together with various CMV or tomato aspermy virus strains not only failed to increase the severity of symptoms in infected tomato plants, but ameliorated them in some instances. Comparisons of the two RNAs by hybridization analysis using 32P-labelled complementary DNA probes, indicate that they have partial nucleotide sequence homology. It is suggested that the difference in their primary structure is reflected in their biological properties.

Protein composition of tomato spotted wilt virus

Abstract Analysis of the protein composition of tomato spotted wilt virus (TSWV), purified by an improved procedure, by polacrylamide gel electrophoresis, revealed three major structural proteins (of MW 84,000, 50,000, and 29,000d) and a minor one of MW 220,000d. The three major proteins constitute about 98% of the total viral protein and all three were shown to be glycoproteins. One of the major proteins (MW 29,000d) and the minor protein were shown to be associated with subviral particles isolated by treatment of virus with the nonionic detergent Nonidet P-40. Only traces of the other two proteins were detected in the subviral particles. Synthesis of virus-induced proteins in TSWV-infected tobacco leaves was studied by labeling infected and healthy tissue with [3H]and [14C]valine, respectively. The labeled tissues were then fractionated into crude subcellular fractions and protein patterns of healthy and infected tissues were compared by coelectrophoresis on polyacrylamide gels. Only one virus-specific protein (of MW 49,000d) was detected in the virus-enriched fractions; this corresponded with the viral structural protein of MW 50,000d.

Stabilization of capsid structure and enhancement of immunogenicity of cucumber mosaic virus (Q strain) by formaldehyde

Abstract Treatment of the Q strain of cucumber mosaic virus (QCMV) with 0.2% formaldehyde for 24 hr at 4 ° in 0.005 M borate buffer, pH 9, stabilizes capsid structure and enhances immunogenicity in mice. Treated virus remained in suspension on mild heating in the presence of electrolyte whereas normal virus precipitated under similar conditions. Protein shells of formaldehyde-treated virus retained their structure during digestion of the viral RNA with pancreatic ribonuclease while those of normal virus degraded rapidly. Formaldehyde-treated capsids retained their structure on exposure to phosphotungstic acid, pH 6.8, during preparation for electron microscopy under conditions which degraded normal virus. It is concluded that the observed changes in the physical properties of QCMV by formaldehyde treatment are the results of cross linking of reactive sites on the adjacent subunits of the protein shells rendering the structural integrity of the capsids less dependent on bonding between the viral RNA and protein. The enhanced immunogenicity of formaldehyde-treated QCMV may be due to ability of the antigen to retain its structural integrity in the tissues of the animal.

Localization of lettuce necrotic yellows virus in host leaf tissue

Abstract Electron microscopic investigation of sections of lettuce necrotic yellows virus (LNYV)-infected Nicotiana glutinosa L. and Lactuca sativa L. (lettuce) leaves showed the presence of particles in the cytoplasm which are absent in healthy material. These particles are of the same diameter (about 52 mμ) as those found in partially purified preparations of the virus. The particles usually occur in bundles enclosed in double membrane-bounded structures. It is suggested that the inner membranes of these structures are associated with the assembly of the particles.

The bipartite genome of red clover necrotic mosaic virus

Purified preparations of red clover necrotic mosaic virus isolated in Australia have been shown to contain three RNA components whose electrophoretic mobilities in polyacrylamide gel electrophoresis indicate molecular weights of 1.5 x 106 (RNA 1), 0.5 x 10(6) (RNA 2), and 0.14 x 10(6) (RNA 3). Comparisons of the RNAs by hybridization analysis with 3H-labeled complementary DNAs synthesized in vitro have established that RNAs 1 and 2 are unique RNA molecular species with little or no sequence homology between them. However, RNA 3 appears to be a complex mixture of breakdown fragments of both RNA 1 and RNA 2. Infectivity experiments with highly purified preparations of RNAs 1 and 2 have demonstrated that both molecules are essential for infectivity.