Replication of Barley Yellow Dwarf Virus Satellite RNA Transcripts in Oat Protoplasts

Silver, S L; Rasochova, Lada; Dinesh-Kumar, S. P.; Miller, W. Allen

doi:10.1006/viro.1994.1036

Cited by 12 publications

(8 citation statements)

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“…To do this, we used modified BYDV-PAV as a vector to express CYDV-RPV genes. BYDV does not support satRPV RNA replication (41). BYDV has significant sequence similarity to CYDV-RPV only in the coat protein and putative movement protein genes (ORF3, ORF4, and ORF5) (28) that are not needed for BYDV RNA replication in protoplasts (30).…”

Section: Resultsmentioning

confidence: 99%

“…The uncloned products of the second PCR were transcribed by using T7 polymerase. The 322-nt gel-purified monomer obtained by self-cleavage (as above) was coelectroporated into oat protoplasts with 50 ng of viral RNA purified from a mixture of CYDV-RPV and BYDV-PAV isolates (41,44). (The CYDV-RPV mixture accumulates to a higher titer than pure CYDV-RPV in the presence of BYDV-PAV, and the mixture supports satRPV RNA as efficiently as does pure CYDV-RPV [41].)…”

Section: Methodsmentioning

confidence: 99%

“…The 322-nt gel-purified monomer obtained by self-cleavage (as above) was coelectroporated into oat protoplasts with 50 ng of viral RNA purified from a mixture of CYDV-RPV and BYDV-PAV isolates (41,44). (The CYDV-RPV mixture accumulates to a higher titer than pure CYDV-RPV in the presence of BYDV-PAV, and the mixture supports satRPV RNA as efficiently as does pure CYDV-RPV [41].) For the second-round inoculations, total RNA was extracted from infected protoplasts (48 h postinfection [hpi]) and was used to coinoculate fresh protoplasts with 50 ng of helper viral RNA.…”

Section: Methodsmentioning

confidence: 99%

“…Oat (Avena sativa cv. Stout) protoplasts were isolated from cell suspension culture (cell line S226 obtained from Howard Rines, U.S. Department of Agriculture, Agricultural Research Service, University of Minnesota) as previously described (41). Protoplasts were electroporated with 50 ng of viral RNA purified from CYDV-RPV virions and 20 ng of gel-purified monomeric satRPV RNA transcript.…”

Section: Methodsmentioning

confidence: 99%

“…Both genera are in the Luteoviridae family (10). BYDV serotypes that remain classified in the genus Luteovirus do not support replication of satRPV RNA or any other known satellite RNA (41).…”

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confidence: 99%

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cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

Song

Miller

2004

J Virol

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Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In Cereal yellow dwarf polerovirus serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a doublehammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper Barley yellow dwarf luteovirus, was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii) cis-acting elements that interact with viral proteins.Satellite RNAs depend on helper viruses for replication, encapsidation, and dissemination among hosts (47, 48). Satellite RNAs that form circles (circular satellite RNAs) (2,20) encode no functional open reading frames (ORFs). Thus, the helper virus and the host must provide all trans-acting factors necessary for replication. Satellite RNAs accumulate to high levels in the presence of a helper virus, despite having no sequence homology with the helper virus RNA. In contrast, viroids are autonomously replicating circular RNA pathogens that do not require a helper virus or virion (2,14,20). Circular satellite RNAs and viroids (4) are the smallest replicating nucleic acids, ranging from 225 to about 450 nucleotides (nt) in length. Thus, they serve as models for understanding the minimum requirements for the replication of genetic information. This can shed light on virus replication processes and lead to means of interfering with virus infection.Numerous studies of replication intermediates and the processing of satellite RNAs support the following rolling circle mechanism for replication of circular satellite RNAs (4, 5). Upon exiting the virion in the cell, the infectious circular plusstrand RNA is copied into a linear, multimeric minus strand (4, 5). (For satellite RNAs that encode no ORFs, we define the encapsidated strand as plus sense.) The minus stran...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

Song

Miller

2004

J Virol

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The Satellite RNA of Barley Yellow Dwarf Virus-RPV Is Supported by Beet Western Yellows Virus in Dicotyledonous Protoplasts and Plants

et al. 1997

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The subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to Capsella bursa-pastoris plants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants.

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