Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability

Abstract

Brome mosaic virus (BMV) RNA replication has been examined in a number of systems, including Saccharomyces cerevisiae. We developed an efficient T-DNA-based gene delivery system using Agrobacterium tumefaciens to transiently express BMV RNAs in Nicotiana benthamiana. The expressed RNAs can systemically infect plants and provide material to extract BMV replicase that can perform template-dependent RNA-dependent RNA synthesis in vitro. We also expressed the four BMV-encoded proteins from nonreplicating RNAs and analyzed their effects on BMV RNA accumulation. The capsid protein that coinfiltrated with constructs expressing RNA1 and RNA2 suppressed minus-strand levels but increased plus-strand RNA accumulation. The replication proteins 1a and 2a could function in trans to replicate and transcribe the BMV RNAs. None of the BMV proteins or RNA could efficiently suppress posttranscriptional silencing. However, 1a expressed in trans will suppress the production of a recombinant green fluorescent protein expressed from the nontranslated portions of BMV RNA1 and RNA2, suggesting that 1a may regulate translation from BMV RNAs. BMV replicase proteins 1a did not affect the accumulation of the BMV RNAs in the absence of RNA replication, unlike the situation reported for S. cerevisiae. This work demonstrates that the Agrobacterium-mediated gene delivery system can be used to study the cis-and trans-acting requirements for BMV RNA replication in plants and that significant differences can exist for BMV RNA replication in different hosts.Successful viral infection requires specific interactions between the viral genomic RNAs and viral proteins (13). The interactions are likely complex since the virus needs to regulate the amount and timing of RNA synthesis and then escape or counteract cellular defenses (44,54,70,74,90,95). We use brome mosaic virus (BMV) as a model system to analyze the interaction of viral proteins and RNAs.BMV belongs to the alphavirus-like superfamily of plant and animal positive-strand RNA viruses. A number of different systems have been used to analyze the requirements for BMV RNA replication and spread. These include the BMV RNA replicase that can direct RNA synthesis in vitro (1, 88), barley and tobacco protoplasts that can be transfected with BMV RNAs (25,35,48), plants that can be used to analyze local and systemic infections (28,57,68), and Saccharomyces cerevisiae, which is permissive for BMV RNA replication and transcription (39). These diverse systems have yielded useful insights into the mechanism of viral infection. Furthermore, the results from different systems are generally in agreement, but some notable differences have been observed (31,65,86).The BMV genome is divided into three capped RNAs, designated RNA1, RNA2, and RNA3 (2). The RNAs make four proteins. RNA1 encodes the multifunctional protein 1a, which methylates and caps BMV RNAs (3, 47) and transports BMV RNAs and the replication-associated proteins to the site of RNA replication (18,24,77,87). RNA2 encodes protein 2a, the RNA-depende...

Selective Repression of Translation by the Brome Mosaic Virus 1a RNA Replication Protein

“…1A). Total RNAs were extracted at 44 h postagroinfiltration, a time at which we could observe robust (Ϫ)-and (ϩ)-strand replication products (14). The expression of BMV MP, the HCV NS3 helicase, and the HCV NS5B polymerase had only modest effects on viral RNA replication, as detected by strand-specific riboprobes complementary to the conserved 3Ј 200 nucleotides of BMV RNAs ( Fig.…”

“…The Agrobacterium-mediated gene delivery system to express the three BMV genomic RNAs for replication and all four of the BMVencoded proteins independent of the viral RNAs was developed by Gopinath et al (14). Constructs that express the hepatitis C virus (HCV) full-length NS3 and NS5B proteins were amplified from the HCV subgenomic replicon (29), using 5Ј NcoI and 3Ј XbaI restriction sites that were built into the primers (the sequences of all primers will be made available upon request).…”

“…Using this system, we made a preliminary observation that a green fluorescent protein (GFP) reporter expressed from BMV RNA1 and RNA2 was repressed by 1a in N. benthamiana (14). 1a was required and sufficient for this activity (14). How this activity relates to other functions of the 1a protein is not clear.…”

“…Recently, an Agrobacterium-mediated T-DNA gene delivery system was established to study BMV replication, gene expression, and RNA packaging in Nicotiana benthamiana (4,14). The agroinfiltration system provides robust transient expression of viral proteins and allows careful dissection of the cisand trans-acting requirements for BMV RNA replication in plants.…”

“…The agroinfiltration system provides robust transient expression of viral proteins and allows careful dissection of the cisand trans-acting requirements for BMV RNA replication in plants. Using this system, we made a preliminary observation that a green fluorescent protein (GFP) reporter expressed from BMV RNA1 and RNA2 was repressed by 1a in N. benthamiana (14). 1a was required and sufficient for this activity (14).…”