Allan R. Gould

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.

Characterization of a satellite RNA associated with cucumber mosaic virus.

Abstract Two types of RNA, each with a molecular weight of approximately 0.12 × 10 6 , designated RNA 5 and satellite RNA, have been found in purified cucumber mosaic virus (CMV) preparations and have been characterized by molecular hybridization analysis using 32 P-labeled complementary DNA probes transcribed from these RNAs. RNA 5 usually makes up about 5% or less by weight of the total viral RNA and was shown to consist of specific cleavage products of CMV RNAs 1–4. Its nucleotide complexity was equivalent to about three times its molecular weight. By contrast, satellite RNA could form up to about 50% by weight of virion RNA and had the following properties: (1) It contained a unique nucleotide sequence with no homology with CMV RNAs, (2) CMV and tomato aspermy virus, but not alfalfa mosaic virus or tobacco ringspot virus, could function as helper viruses for its replication and encapsidation, (3) its nucleotide sequence was independent of the host plant and the helper virus used for its propagation and it was not derived from a host plant RNA, and (4) it was not a negative copy of any of the CMV RNA species. We concluded that this RNA is a true satellite RNA and has no relationship to the RNA found in defective interfering particles of animal viruses.

Citrus exocortis viroid: nucleotide sequence and secondary structure of an Australian isolate

Exocortis or ‘scaly butt disease’ of citrus is distributed world-wide [I]. The causative agent is citrus exocortis viroid (CEV) [2,3] which is a member of that unique group of plant pathogens, the viroids, of which only 8 have been described [ 1,4,5]. Like other members of the group, CEV consists of a singlestranded covalently closed circular RNA molecule which is highly base-paired, rod-like, infectious and non-encapsidated [ 1,4]. The primary sequence and proposed secondary structure of only 3 viroids have been reported so far: potato spindle tuber viroid (PSTV) with 359 residues [6]; chrysanthemum stunt viroid (CSV) with 356 residues [7]; and avocado sunblotch viroid (ASBV) with 247 residues [8]. We report here the sequence and proposed secondary structure of the 371 residues of an Australian isolate of CEV and discuss the significance of the extensive sequence homology which exists between PSTV, CSV and CEV.

Oligomers of avocado sunblotch viroid are found in infected avocado leaves

The most abundant form of avocado sunblotch viroid (ASBV) in extracts of infected leaves of avocado (Persea americana) is a single-stranded covalent RNA circle [l] of 247 residues [2]. Highly purified preparations of the circular viroid infect avocado and induce the sunblotch disease [3]. ASBV has a limited sequence homology with 3 other viroids which have extensive sequence homology with each other [2,4]; potato spindle tuber viroid (PSTV), chrysanthemum stunt viroid (CSV) and citrus exocortis viroid (CEV). The level of ASBV in nucleic acid extracts from leaves of infected avocado leaves from different trees varied over a 10 OOO-fold range [5], while there was a lOOO-fold variation in the concentration of ASBV in extracts prepared from 6 branches of the same tree [6]. We report here that extracts of leaf tissue from strongly infected avocado seedlings contain an oligomeric series of RNAs which are integral multiples of the unit length ASBV and are of the same (+) polarity. Complementary (-) RNAs are also present but in much lower concentrations.

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.

Studies on encapsidated viroid-like RNA II. Purification and characterization of a viroid-like RNA associated with velvet tobacco mottle virus (VTMoV)

A viroid-like RNA has been purified from velvet tobacco mottle virus (VTMoV) capsids. This RNA exists as a covalently closed circular molecule of RNA as well as in a linear form with a molecular weight of approximately 1.2 x 10(5). Complementary DNA (cDNA) analysis indicates that the circular and linear forms of the viroid-like RNA contain indistinguishable nucleotide sequences and are unique RNA species. The viroid-like RNA has no nucleotide sequence homology with any other encapsidated RNAs and exhibits an absorbance-temperature profile, as well as nuclease sensitivities, typical of a viroid. The three other RNAs encapsidated in VTMoV virions were also investigated by cDNA analysis. Two of these (designated RNAs 1a and 1b, with molecular weights of 0.63 x 10(6) and 0.25 x 10(6), respectively) are shown to be breakdown fragments of the largest RNA encapsidated by VTMoV (designated RNA 1) with a molecular weight of 1.5 x 10(6).

Studies on encapsidated viroid-like RNA. III. Comparative studies on RNAs isolated from velvet tobacco mottle virus and Solanum nodiflorum mottle virus.

Solanum nodiforum mottle virus (SNMV) encapsidates two unique RNA species as shown by 3H-complementary DNA hybridization analysis. They are RNA 1, a long linear single-stranded molecule with a molecular weight of 1.5 x 10(6) and RNA 2 and RNA 3 which are circular and linear forms of the same molecule, respectively. Electron microscopic examination and thermal denaturation studies indicate a viroid-like structure for RNA 2 of SNMV. Electrophoretic analysis of the viroid-like RNA encapsidated by SNMV indicates it has a slightly larger molecular weight (0.13 x 108) than the viroid-like RNA recently isolated from velvet tobacco mottle virus (VTMoV). Complementary DNA (cDNA) analysis revealed that SNMV RNA 2 contains the entire nucleotide sequence of VTMoV RNA 2, plus an additional 40-50 nucleotides not present in the latter. Comparative sequence homology studies between the large RNAs encapsidated by SNMV and VTMoV (both of MW 1.5 x 10(6)) revealed only partial homology (20%). However, the level of S1 nuclease resistance could be increased by the presence of higher salt concentrations during hybridization and subsequent S1 nuclease assay. This indicates that many smaller regions of homology are present between VTMoV RNA 1 and SNMV RNA 1. This is the second example of an encapsidated viroid-like RNA.

Studies on encapsidated viroid-like RNA IV. requirement for infectivity and specificity of two RNA components from velvet tobacco mottle virus

Neither the virus-like RNA (RNA 1) nor the viroid-like RNA (RNA 2) of velvet tobacco mottle virus (VTMoV) is capable of independent replication in Nicotiana clevelandii. The function of RNA 2 for the RNA 1 replication could not be fulfilled by conventional viroids such as those of chrysanthemum stunt, citrus exocortis, or the viroid-like RNAs associated with avocado sunblotch and cadang-cadang disease of coconut. Similarly, RNA 2 failed to replicate when coinoculated with RNAs of conventional viruses such as those of southern bean mosaic, sowbane mosaic, galinsoga mosaic, red clover necrotic mosaic, and carnation mottle, all of which possess genomes with molecular weights similar to those of VTMoV RNA 1. The interdependence of the two VTMoV RNAs for biological activity was so highly specific that the function of VTMoV RNA 2 could not be replaced by the viroid-like RNA 2 from solanum nodiflorum mottle virus (SNMV), a closely related virus, for the replication of VTMoV RNA 1. Similarly, SNMV RNA 2 could not be replaced by VTMoV RNA 2 for the replication of SNMV RNA 1. These data support the view that RNA 2 cannot be a satellite RNA or an independent viroid. It seems that viruses such as VTMoV may have originated from associations of conventional single-stranded RNA viruses and viroids or, alternatively, may represent a stage in the evolution of viroids from viruses.

Accumulation of messenger RNA for extracellular enzymes as a general phenomenon in Bacillus amyloliquefaciens

Abstract Recently it has been established that in harvested Bacillus amyloliquefaciens there exists a pool of messenger RNA which can support de novo extracellular protease production for 80 minutes Both, McInnes, Hanlon, May & Elliott 1972. The present study shows that this is not peculiar to protease; the other two extracellular enzymes under study, α-amylase and ribonuclease, show similar characteristics. Synthesis of all three extracellular enzymes is insensitive to rifampicin and actinomycin D for extended time periods after cessation of RNA and cellular protein synthesis.

Studies on the protoplast-bursting factor from Bacillus amyloliquefaciens

Previous studies in this laboratory [l] have shown that Bacillus amyloliquefaciens secretes a heat stable factor into the external medium which has antibiotic activity towards many gram positive organisms and which is capable of rapidly lysing protoplasts of Bacillus amyloliquefaciens. This factor is a peptidelipid, the structure of which appears to be identical to that for ‘Surfactin’ isolated from Bacillus subtilis supernatant by Arima et al. [2]. The structure shown in scheme 1 was proposed by Kakinuma et al. [3] , and was the basis for building a space filling model. It has now been shown that the biological activity depends upon the integrity of the lactone bond and that hydrolysis or reversion to the closed ring form can be simply attained by treatment with alkaline or acidic conditions.