Studies on Functional Interaction between Brome Mosaic Virus Replicase Proteins during RNA Recombination, Using Combined Mutants in Vivo and in Vitro

ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

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Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

Journal of Virology ◽

10.1128/jvi.78.12.6091-6101.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6091-6101 ◽

Cited By ~ 19

Author(s):

K. Sivakumaran ◽

Seung-Kook Choi ◽

Masarapu Hema ◽

C. Cheng Kao

Keyword(s):

Mosaic Virus ◽

Rna Synthesis ◽

Core Promoter ◽

Brome Mosaic Virus ◽

Wild Type ◽

Subgenomic Rna ◽

The Core ◽

Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

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In Vivo Packaging of Brome Mosaic Virus RNA3, but Not RNAs 1 and 2, Is Dependent on a cis-Acting 3′ tRNA-Like Structure

Journal of Virology ◽

10.1128/jvi.01500-06 ◽

2006 ◽

Vol 81 (1) ◽

pp. 173-181 ◽

Cited By ~ 24

Author(s):

Padmanaban Annamalai ◽

A. L. N. Rao

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Untranslated Region ◽

Northern Blot Analysis ◽

Brome Mosaic Virus ◽

Wild Type ◽

Cis Acting

ABSTRACT The four encapsidated RNAs of brome mosaic virus (BMV; B1, B2, B3, and B4) contain a highly conserved 3′ 200-nucleotide (nt) region encompassing the tRNA-like structure (TLS) which is required for packaging in vitro (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA 99:655-660, 2002). To validate these observations in vivo, we performed packaging assays using Agrobacterium-mediated transient expression of RNAs and coat protein (CP) (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005). Coexpression of TLS-less constructs of B1 or B2 or B3 and CP mRNAs in Nicotiana benthamiana leaves resulted in packaging of TLS-less B1 and B2 but not B3, suggesting that packaging of B3 requires the TLS in cis. This conjecture was confirmed by the efficient packaging of a B3 chimera in which the viral TLS was replaced with a cellular tRNATyr. When N. benthamiana leaves were infiltrated with a mixture of transformants containing wild-type B1 (wtB1) plus wtB2 plus a TLS-less B3 (wtB1+wtB2+TLS-lessB3), the 3′ end of progeny B3 was restored by heterologous recombination with that of either B1 or B2. This intrinsic cis-requirement of TLS in promoting B3 packaging was further confirmed when a mixture containing agrotransformants of TLS-less B1+B2+B3 was supplemented with either wtB4 or a 3′ 200-nt or 3′ 336-nt untranslated region (UTR) of B3. Northern blot analysis followed by sequencing of B3 progeny revealed that replication of TLS-less B3, but not TLS-less B1 or B2, was fully restored due to recombination with TLS from transiently expressed wtB4 or the B3 3′ UTR. Collectively, these observations suggested that the requirement of a cis-acting TLS is distinct for B3 compared with B1 or B2.

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Packaging of Brome Mosaic Virus Subgenomic RNA Is Functionally Coupled to Replication-Dependent Transcription and Translation of Coat Protein

Journal of Virology ◽

10.1128/jvi.01186-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10096-10108 ◽

Cited By ~ 55

Author(s):

Padmanaban Annamalai ◽

A. L. N. Rao

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Expression System ◽

Brome Mosaic Virus ◽

Flock House Virus ◽

Rna Packaging ◽

Reading Frame ◽

Positive Strand Rna

ABSTRACT In Brome mosaic virus (BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an Agrobacterium-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4Trans and CPTrans) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4Rep and CPRep), were mixed in all four pair-wise combinations and infiltrated to Nicotiana benthamiana leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus).

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Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo

Nucleic Acids Research ◽

10.1093/nar/gkn896 ◽

2008 ◽

Vol 36 (22) ◽

pp. 7230-7239 ◽

Cited By ~ 14

Author(s):

R. A. Karran ◽

K. A. Hudak

Keyword(s):

Viral Replication ◽

Mosaic Virus ◽

Intergenic Region ◽

Brome Mosaic Virus

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The AU-Rich RNA Recombination Hot Spot Sequence of Brome Mosaic Virus Is Functional in Tombusviruses: Implications for the Mechanism of RNA Recombination

Journal of Virology ◽

10.1128/jvi.78.5.2288-2300.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2288-2300 ◽

Cited By ~ 60

Author(s):

Natalia Shapka ◽

Peter D. Nagy

Keyword(s):

Mosaic Virus ◽

Hot Spots ◽

Hot Spot ◽

Brome Mosaic Virus ◽

Template Switching ◽

Rna Recombination ◽

Replicase Proteins ◽

Recombination Hot Spots ◽

Recombination Junction ◽

Selection Of

ABSTRACT RNA recombination can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase “landing” during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.

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Efficient and Specific Initiation of Subgenomic RNA Synthesis by Cucumber Mosaic Virus Replicase In Vitro Requires an Upstream RNA Stem-Loop

Journal of Virology ◽

10.1128/jvi.74.23.11201-11209.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11201-11209 ◽

Cited By ~ 18

Author(s):

M.-H. Chen ◽

M. J. Roossinck ◽

C. C. Kao

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Synthesis ◽

Core Promoter ◽

Brome Mosaic Virus ◽

Stem Loop ◽

Promoter Sequences ◽

Promoter Recognition

ABSTRACT We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions −28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions −28 to −5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.

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Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination

Journal of Virology ◽

10.1128/jvi.78.16.8552-8564.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8552-8564 ◽

Cited By ~ 16

Author(s):

Rafal Wierzchoslawski ◽

Aleksandra Dzianott ◽

Jozef Bujarski

Keyword(s):

Mosaic Virus ◽

Transcription Initiation ◽

De Novo ◽

Brome Mosaic Virus ◽

Rna Recombination ◽

Recombination Activity ◽

Nucleotide Substitutions ◽

Stem Loop ◽

Subgenomic Promoter

ABSTRACT Previously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3 (A. Bruyere et al., J. Virol. 74:4214-4219, 2000; R. Wierzchoslawski et al., J. Virol. 77:6769-6776, 2003). In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the +1 or +2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.

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Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

Journal of Virology ◽

10.1128/jvi.75.4.1879-1887.2001 ◽

2001 ◽

Vol 75 (4) ◽

pp. 1879-1887 ◽

Cited By ~ 54

Author(s):

Maurice W. Van Der Heijden ◽

Jan E. Carette ◽

Poula J. Reinhoud ◽

Anita Haegi ◽

John F. Bol

Keyword(s):

Mosaic Virus ◽

Alfalfa Mosaic Virus ◽

Polymerase Activity ◽

Brome Mosaic Virus ◽

Helicase Domain ◽

Yeast Two Hybrid System ◽

Replicase Proteins ◽

The Family ◽

Rna Polymerase Activity

ABSTRACT Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908–8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.

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Towards plant resistance to viruses using protein-only RNase P

Nature Communications ◽

10.1038/s41467-021-21338-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anthony Gobert ◽

Yifat Quan ◽

Mathilde Arrivé ◽

Florent Waltz ◽

Nathalie Da Silva ◽

...

Keyword(s):

Mosaic Virus ◽

Virus Replication ◽

Yield Loss ◽

Rnase P ◽

Plant Viruses ◽

Yellow Mosaic Virus ◽

Resistance To Viruses ◽

Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

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