scholarly journals cis-Acting Elements Required for Efficient Packaging of Brome Mosaic Virus RNA3 in Barley Protoplasts

2003 ◽  
Vol 77 (18) ◽  
pp. 9979-9986 ◽  
Author(s):  
Tri Asmira Damayanti ◽  
Satoshi Tsukaguchi ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Brome Mosaic Virus ◽  
Proximal Region ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Reading Frame ◽  
Cis Acting ◽  
Infected Cells ◽  
Packaging Efficiency

ABSTRACT Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3′-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3′-proximal region of the CMV 3a ORF in B3Cmp with the 3′-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3′-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

scholarly journals Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

2019 ◽  
Author(s):  
Antara Chakravarty ◽  
Christian Beren ◽  
Rees Garmann ◽  
A.L.N. Rao
Keyword(s):  
Mosaic Virus ◽  
Transient Expression ◽  
Rna Binding ◽  
Conformational Dynamics ◽  
Brome Mosaic Virus ◽  
Crystallographic Structure ◽  
Binding Motif ◽  
Genomic Rnas ◽  
Viral Capsids

ABSTRACTViral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replicated three-molecule RNA progeny of Brome mosaic virus (BMV) are packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Type 1 (B1v) and type 2 (B2v) virions respectively package genomic RNA1 or RNA2, while type 3 (B3+4v) co-packages genomic RNA3 (B3) and its sub-genomic RNA4 (B4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Physical and biochemical approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types, so protease-based mapping experiments were used to analyze the conformational dynamics of the individual virions. The crystallographic structure of the BMV capsid shows four trypsin-cleavage sites (K65, R103, K111 and K165 on the A, B and C subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1v and B2v released only two peptides involving amino acids 2-8 and 16-22 from the N-proximal arginine-rich RNA binding motif. In contrast, B3+4v capsids are unstable to trypsin, releasing several peptides in addition to the four sites predicted to be exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCEThe majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that in solution virions are highly dynamic assemblies. The three genomic RNAs (RNAs 1, 2 and 3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the capsid dynamics by MALDI-TOF analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNAs1 and 2 are more stable and dynamically distinct from those co-packaging RNA3 and 4, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

scholarly journals Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

2005 ◽  
Vol 79 (14) ◽  
pp. 9046-9053 ◽  
Author(s):  
Jen-Wen Lin ◽  
Hsiao-Ning Chiu ◽  
I-Hsuan Chen ◽  
Tzu-Chi Chen ◽  
Yau-Heiu Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Minus Strand ◽  
Internal Deletion ◽  
Stem Loop ◽  
Cis Acting ◽  
Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

scholarly journals Brome Mosaic Virus Protein 1a Recruits Viral RNA2 to RNA Replication through a 5′ Proximal RNA2 Signal

2001 ◽  
Vol 75 (7) ◽  
pp. 3207-3219 ◽  
Author(s):  
Jianbo Chen ◽  
Amine Noueiry ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Protein ◽  
Membrane Association ◽  
Stem Loop ◽  
Induced Membrane ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5′-proximal third of RNA2. The RNA2 5′ untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5′-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TΨC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

scholarly journals Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts

2003 ◽  
Vol 77 (10) ◽  
pp. 5703-5711 ◽  
Author(s):  
K. Sivakumaran ◽  
M. Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Previous Analysis ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Stem Loop ◽  
Virus Rna ◽  
Different Levels

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.

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

2006 ◽  
Vol 81 (1) ◽  
pp. 173-181 ◽  
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.

scholarly journals An Alternate Pathway for Recruiting Template RNA to the Brome Mosaic Virus RNA Replication Complex

2003 ◽  
Vol 77 (4) ◽  
pp. 2568-2577 ◽  
Author(s):  
Jianbo Chen ◽  
Amine Noueiry ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Terminal Region ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Stem Loop ◽  
Reading Frame ◽  
Replication Complex ◽  
Alternate Pathway

ABSTRACT The multidomain RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, plays key roles in assembly and function of the viral RNA replication complex. 1a, which encodes RNA capping and helicase-like domains, localizes to endoplasmic reticulum membranes, recruits BMV 2a polymerase and viral RNA templates, and forms membrane-bound, capsid-like spherules in which RNA replication occurs. cis-acting signals necessary and sufficient for RNA recruitment by 1a have been mapped in BMV genomic RNA2 and RNA3. Both signals comprise an extended stem-loop whose apex matches the conserved sequence and structure of the TΨC stem-loop in tRNAs (box B). Mutations show that this box B motif is crucial to 1a responsiveness of wild-type RNA2 and RNA3. We report here that, unexpectedly, some chimeric mRNAs expressing the 2a polymerase open reading frame from RNA2 were recruited by 1a to the replication complex and served as templates for negative-strand RNA synthesis, despite lacking the normally essential, box B-containing 5′ signal. Further studies showed that this template recruitment required high-efficiency translation of the RNA templates. Moreover, multiple small frameshifting insertion or deletion mutations throughout the N-terminal region of the open reading frame inhibited this template recruitment, while an in-frame insertion did not. Providing 2a in trans did not restore template recruitment of RNAs with frameshift mutations. Only those deletions in the N-terminal region of 2a that abolished 2a interaction with 1a abolished template recruitment of the RNA. These and other results indicate that this alternate pathway for 1a-dependent RNA recruitment involves 1a interaction with the translating mRNA via the 1a-interactive N-terminal region of the nascent 2a polypeptide. Interaction with nascent 2a also may be involved in 1a recruitment of 2a polymerase to membranes.

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

2006 ◽  
Vol 80 (20) ◽  
pp. 10096-10108 ◽  
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).

scholarly journals Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Antara Chakravarty ◽  
Vijay S. Reddy ◽  
A. L. N. Rao
Keyword(s):  
Mosaic Virus ◽  
Transient Expression ◽  
Rna Binding ◽  
Conformational Dynamics ◽  
Brome Mosaic Virus ◽  
Crystallographic Structure ◽  
Binding Motif ◽  
Genomic Rnas ◽  
Viral Capsids

ABSTRACT Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

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

2000 ◽  
Vol 74 (23) ◽  
pp. 11201-11209 ◽  
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.

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

2004 ◽  
Vol 78 (16) ◽  
pp. 8552-8564 ◽  
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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