Self-cleaving circular RNA associated with rice yellow mottle virus is the smallest viroid-like RNA.

We report the sequence, structural features, and self-cleaving activity of the small circular RNA (sc-RNA) associated with rice yellow mottle sobemovirus (RYMV). At 220 nucleotides, the RYMV sc-RNA represents the smallest naturally occurring viroid-like RNA currently documented in the literature. It is similar to other circular satellite RNAs (sat-RNAs) and viroids in being G-C-rich with a high level of self-complementarity. The predicted native structure is essentially a rod with one branched terminus. A region of the RYMV sc-RNA, constituting 24% of the sequence, exhibits 89% identity to the sat-RNA associated with the Australasian isolates of lucerne transient streak sobemovirus. This region is also structurally similar in all three RNAs in that it forms the left terminus of each rod. Dimeric runoff transcripts of cloned RYMV sc-RNA undergo efficient autocatalytic in vitro cleavage in the (+) but not the (-) polarity. Analysis of the (+) sequence indicates the presence of a hammerhead ribozyme resembling that of carnation small retroviroid-like RNA and the genomic satellite transcript of newt. Inefficient cleavage of (+) monomeric transcripts, and a short stem III in the hammerhead, are features consistent with a double-hammerhead mode of self-cleavage. The presence of sat-RNA and retroviroid-like structures within a single RNA suggests a possible role for the RYMV sc-RNA as an evolutionary intermediate between these subviral RNAs.

Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

Pathogenesis-related proteins in lima bean leaves infected with tobacco ringspot virus and their distribution within and around local lesions.

Tobacco ringspot virus (TRSV) induces circular, darkbrown local lesions on primary leaves of lima bean (Phaseolus lunatus cv Nemagreen) with a concomitant production of three basic and three acidic pathogenesisrelated (PR) proteins. The three basic proteins are: a 21 kDa protein related serologically to Pinto bean PR-4d and tobacco PR-5 proteins; a 36 kDa glucanase that is related to tobacco PR-2; and, a 31 kDa chitinase related serologically to ethylene-induced bean chitinase. The three acidic 18 kDa lima bean PR proteins are serologically similar and probably are charged isomers of the same protein. The 21 kDa basic protein and the 18 kDa acidic protein accumulated preferentially at the lesion center while the 31 kDa chitinase and TRSV were distributed evenly throughout the necrotic area. In green tissue immediately surrounding a lesion, the amounts of PR proteins were comparable to or lower than those in the necrotic area, and virions were not detected. This mode of spatial distribution indicates that lima bean PR proteins are not involved in TRSV localization, and is consistent with other observations that PR proteins play no direct role in restricting viral spread.

Temperature-induced structural stabilization of the encapsidated RNA of southern bean mosaic virus and its biologic significance.

Virions of southern bean mosaic virus (SBMV) show two distinct sensitivity patterns upon heating. Infectivity loss and capsid denaturation occur concurrently if virions are exposed at 50-55 degrees in 0.1 M sodium phosphate buffer, pH 7.5, or 0.1 M glycine-phosphate buffer, pH 9.0. Contrastingly, virions are inactivated without any detectable capsid damage at 65-70 degrees in 0.1 M sodium phosphate buffer, pH 6.0, 0.1 M Tris-HCI buffer, pH 7.5, or 0.1 M glycine-NaOH buffer, pH 9.0. Heat treatment causes no genomic degradation in these two situations; the divergent sensitivity of virions is due, apparently, to a differential thermal tolerance of the capsid protein to the buffer components and/or pH. SBMV-RNA isolated from virions inactivated at 65 degrees in 0.1 M Tris-HCl buffer, pH 7.5, possesses low infectivity (1-2%). Observations based upon sucrose gradient sedimentation, temperature: absorbance relationship, and sensitivity to ribonuclease T(1) suggest that such RNA is structurally more compact and stable relative to that of the RNA from the nonheated virions. Neither the capsid protein nor the genome protein plays a direct role in the temperature-induced structural stabilization of SBMV-RNA in situ. If treated with 8 M urea, 50% formamide or exposed at 55 degrees , the infectivity of RNA from the heat-inactivated SBMV is restored and is comparable to that of the RNA isolated from the nonheated virions. The observed mode of SBMV thermal inactivation, i.e., stabilization of RNA structure in situ, is unique among the viruses. Furthermore, these results suggest that the ability to initiate infection depends upon the secondary structure of the SBMV genome.

pH-Dependent urea sensitivity of southern bean mosaic virus.

The behavior of southern bean mosaic virus (SBMV) virions in urea was markedly pH dependent and revealed several novel features. In the absence of urea (5 degrees , 1 hr) SBMV was stable as a 115 S entity between pH 2.5 and 9.5, yielded a slowly sedimenting (ca 60-65 S) entity at pH 10.5, and was degraded into a 35-40 S product at pH 11.0. In 4.0 M urea, SBMV was precipitated irreversibly at pH 2.5 or 10.0, yielded variously sedimenting aggregates at pH 3.0, was transformed progressively into a 80 S entity between pH 7.0 and 9.5, or remained structurally intact at pH 3.5 to 6.5. In 8.O M urea, SBMV was precipitated at pH 3.5 and below or pH 7.5 and above, showed a tendency for the particle:particle interaction at pH 6.5 and 7.0, but remained stable between pH 4.0 and 6.0. Under conditions where SBMV was somewhat destabilized with urea, increase in the temperature or ionic concentration caused virion precipitation without any detectable degradation. SBMV was dissociated with 4.0 or 8.0 M urea at pH 5.5 but only in the presence of 1.0 M NaCl and at the elevated temperatures (>50 degrees ). These results on the rather unusual SBMV behavior in urea, as compared to other viruses, are discussed in relation to the available information on the SBMV stabilizing interactions.

Efficiency of nitrous acid as an inactivating and mutagenic agent of intact tobacco mosaic virus and its isolated nucleic acid.

When tobacco mosaic virus (TMV) and its isolated nucleic acid (TMV-RNA) were treated with nitrous acid, the nucleic acid was inactivated six times faster than the intact virus. Inactivation of both the infectious entities was exponential with treatment time to 0.1% level of survival. Eight different mutant phenotypes were scored after inactivation of TMV and TMV-RNA to 50, 10, 1.0, and 0.1% survival levels. Significantly more mutants in relation to unaltered isolates were induced at all levels of survival upon nitrous acid treatment of TMV than of TMV-RNA. Furthermore, the proportion of two specific mutant phenotypes was significantly greater in treated TMV than in treated TMV-RNA. No qualitative differences, however, were observed between the mutational spectra of nitrous acid-treated TMV and TMV-RNA. These results indicate that, in the intact virus, the viral capsid protects some of the sites involved in lethality; thus, proportionately more mutants are induced on nitrous acid treatment of TMV versus TMV-RNA.