Identification and functional characterization of regions that can be crosslinked to RNA in the helicase-like domain of BaMV replicase

Han, Yu-Tsung; Hsu, Yau‐Heiu; Lo, Chia-Wen; Meng, Menghsiao

doi:10.1016/j.virol.2009.04.013

Cited by 10 publications

(7 citation statements)

References 40 publications

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“…The viral protein was expressed as inclusion bodies in E. coli BL21(DE3). To purify the viral protein, inclusion bodies were dissolved and refolded according to a protocol described previously (10). In detail, inclusion bodies were dissolved in protein buffer that contained 50 mM Tris (pH 7.5), 20 mM KCl, 0.1% Brij-35, 10% glycerol, and 10 mM ␤-mercaptoethanol supplemented with 8 M urea.…”

Section: Methodsmentioning

confidence: 99%

The Interaction between Bamboo Mosaic Virus Replication Protein and Coat Protein Is Critical for Virus Movement in Plant Hosts

Lee

et al. 2011

J Virol

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Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.To spread throughout hosts, plant viruses have evolved a number of pathways to allow their progeny to pass across plasmodesmata into neighboring cells and travel along the vascular system (8, 26). The virus-encoded movement proteins play a pivotal role through diverse mechanisms in these cellto-cell and vascular transports. Ancillary proteins, for example, the viral coat proteins (CPs) in some cases, and host factors may also participate in these processes. Numerous studies have been conducted to elucidate the movement mechanisms. Many of the results have been summarized in a number of recent reviews (23,28,30). They provided in-depth discussions on issues such as the identification and characterization of the involved viral and host proteins and the transport models for some exemplified viruses, such as Tobacco mosaic virus (TMV) and Potato virus X (PVX). Despite these efforts, many details of the processes remain elusive.Members of the genus Potexvirus have a positive-strand RNA genome that contains five open reading frames (ORFs), a 5Ј methyl cap, and a 3Ј poly(A) tail. ORF1 encodes the viral replication protein, consisting of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain (RdRp) from the N terminus to the C terminus (16,17). The HLD has RNA 5Ј-triphosphatase and nucleoside triphosphatase (NTPase) activities (18). With the co...

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Section: Methodsmentioning

confidence: 99%

The Interaction between Bamboo Mosaic Virus Replication Protein and Coat Protein Is Critical for Virus Movement in Plant Hosts

Lee

et al. 2011

J Virol

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“…The method for the cross‐linking of protein and biotinylated RNA by formaldehyde was based on a previous description (Han et al ., ). Formaldehyde is a reversible cross‐linking agent widely used in protein–DNA, protein–RNA and protein–protein interactions (Niranjanakumari et al ., ; Perez‐Romero and Imperiale, ; Sutherland et al ., ).…”

Section: Methodsmentioning

confidence: 99%

Two key arginine residues in the coat protein of Bamboo mosaic virus differentially affect the accumulation of viral genomic and subgenomic RNAs

Hung

Lin

et al. 2013

Molecular Plant Pathology

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The interactions between viral RNAs and coat proteins (CPs) are critical for the efficient completion of infection cycles of RNA viruses. However, the specificity of the interactions between CPs and genomic or subgenomic RNAs remains poorly understood. In this study, Bamboo mosaic virus (BaMV) was used to analyse such interactions. Using reversible formaldehyde cross-linking and mass spectrometry, two regions in CP, each containing a basic amino acid (R99 and R227, respectively), were identified to bind directly to the 5' untranslated region of BaMV genomic RNA. Analyses of the alanine mutations of R99 and R227 revealed that the secondary structures of CP were not affected significantly, whereas the accumulation of BaMV genomic, but not subgenomic, RNA was severely decreased at 24 h post-inoculation in the inoculated protoplasts. In the absence of CP, the accumulation levels of genomic and subgenomic RNAs were decreased to 1.1%-1.5% and 33%-40% of that of the wild-type (wt), respectively, in inoculated leaves at 5 days post-inoculation (dpi). In contrast, in the presence of mutant CPs, the genomic RNAs remained about 1% of that of wt, whereas the subgenomic RNAs accumulated to at least 87%, suggesting that CP might increase the accumulation of subgenomic RNAs. The mutations also restricted viral movement and virion formation in Nicotiana benthamiana leaves at 5 dpi. These results demonstrate that R99 and R227 of CP play crucial roles in the accumulation, movement and virion formation of BaMV RNAs, and indicate that genomic and subgenomic RNAs interact differently with BaMV CP.

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“…Nonetheless, the greater value of K m (ATP) than K m (RNA) , which is about 2.5 fold, suggests that more active-site residues are involved in RNA binding. The peptidyl regions employed by the HLD to bind biotinylated RNA were mapped by the reversible formaldehyde crosslinking method followed by tandem mass spectrometry (Han et al, 2009). Five peptidyl regions were identified.…”

Section: Helicase-like Domain (Hld)mentioning

confidence: 99%

“…Compared with the well-characterized members in SF1, e.g., DNA helicase PcrA, the BaMV HLD seems to bind RNA using a different set of peptidyl regions. Mutagenesis of positively charged residues in these regions showed that some residues, e.g., K603 and R628, have a role in the virus movement (Han et al, 2009). …”

Section: Helicase-like Domain (Hld)mentioning

confidence: 99%

Function and Structural Organization of the Replication Protein of Bamboo mosaic virus

Meng

Lee

2017

Front. Microbiol.

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The genus Potexvirus is one of the eight genera belonging to the family Alphaflexiviridae according to the Virus Taxonomy 2015 released by International Committee on Taxonomy of Viruses (www.ictvonline.org/index.asp). Currently, the genus contains 35 known species including many agricultural important viruses, e.g., Potato virus X (PVX). Members of this genus are characterized by flexuous, filamentous virions of 13 nm in diameter and 470–580 nm in length. A potexvirus has a monopartite positive-strand RNA genome, encoding five open-reading frames (ORFs), with a cap structure at the 5′ end and a poly(A) tail at the 3′ end. Besides PVX, Bamboo mosaic virus (BaMV) is another potexvirus that has received intensive attention due to the wealth of knowledge on the molecular biology of the virus. In this review, we discuss the enzymatic activities associated with each of the functional domains of the BaMV replication protein, a 155-kDa polypeptide encoded by ORF1. The unique cap formation mechanism, which may be conserved across the alphavirus superfamily, is particularly addressed. The recently identified interactions between the replication protein and the plant host factors are also described.

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Identification and functional characterization of regions that can be crosslinked to RNA in the helicase-like domain of BaMV replicase

Cited by 10 publications

References 40 publications

The Interaction between Bamboo Mosaic Virus Replication Protein and Coat Protein Is Critical for Virus Movement in Plant Hosts

The Interaction between Bamboo Mosaic Virus Replication Protein and Coat Protein Is Critical for Virus Movement in Plant Hosts

Two key arginine residues in the coat protein of Bamboo mosaic virus differentially affect the accumulation of viral genomic and subgenomic RNAs

Function and Structural Organization of the Replication Protein of Bamboo mosaic virus

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