Effects of Modification of the Transcription Initiation Site Context on Citrus Tristeza Virus Subgenomic RNA Synthesis

ABSTRACT Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3′-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5′ termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5′ or 3′ of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

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A Non-Conserved p33 Protein of Citrus Tristeza Virus Interacts with Multiple Viral Partners

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-11-19-0328-fi ◽

2020 ◽

Vol 33 (6) ◽

pp. 859-870 ◽

Cited By ~ 2

Author(s):

Thi Nguyet Minh Dao ◽

Sung-Hwan Kang ◽

Aurélie Bak ◽

Svetlana Y. Folimonova

Keyword(s):

Host Range ◽

Citrus Tristeza Virus ◽

Protein A ◽

Integral Membrane Protein ◽

Open Reading Frames ◽

Bimolecular Fluorescence Complementation ◽

In Planta ◽

Tristeza Virus ◽

Citrus Tristeza

The RNA genome of citrus tristeza virus (CTV), one of the most damaging viral pathogens of citrus, contains 12 open reading frames resulting in production of at least 19 proteins. Previous studies on the intraviral interactome of CTV revealed self-interaction of the viral RNA-dependent RNA polymerase, the major coat protein (CP), p20, p23, and p33 proteins, while heterologous interactions between the CTV proteins have not been characterized. In this work, we examined interactions between the p33 protein, a nonconserved protein of CTV, which performs multiple functions in the virus infection cycle and is needed for virus ability to infect the extended host range, with other CTV proteins shown to mediate virus interactions with its plant hosts. Using yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays, we demonstrated that p33 interacts with three viral proteins, i.e., CP, p20, and p23, in vivo and in planta. Coexpression of p33, which is an integral membrane protein, resulted in a shift in the localization of the p20 and p23 proteins toward the subcellular crude-membrane fraction. Upon CTV infection, the four proteins colocalized in the CTV replication factories. In addition, three of them, CP, p20, and p23, were found in the p33-formed membranous structures. Using bioinformatic analyses and mutagenesis, we found that the N-terminus of p33 is involved in the interactions with all three protein partners. A potential role of these interactions in virus ability to infect the extended host range is discussed.

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Transcription Strategy in a Closterovirus: a Novel 5′-Proximal Controller Element of Citrus Tristeza Virus Produces 5′- and 3′-Terminal Subgenomic RNAs and Differs from 3′ Open Reading Frame Controller Elements

Journal of Virology ◽

10.1128/jvi.77.1.340-352.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 340-352 ◽

Cited By ~ 21

Author(s):

Siddarame Gowda ◽

María A. Ayllón ◽

Tatineni Satyanarayana ◽

Moshe Bar-Joseph ◽

William O. Dawson

Keyword(s):

Promoter Activity ◽

Citrus Tristeza Virus ◽

Open Reading Frames ◽

Stem Loop ◽

Reading Frame ◽

Subgenomic Rnas ◽

Modular Evolution ◽

Different Origins ◽

Tristeza Virus ◽

Citrus Tristeza

ABSTRACT Citrus tristeza virus (CTV) produces more than thirty 3′- or 5′-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5′-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3′ genes deleted produce only the larger LMT1 RNAs. These 5′-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5′TR) surrounding the putative termination sites (nt ∼550 to 1000) was duplicated in the 3′ end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5′-terminal plus-strand sgRNA, here much larger (∼11 kb), apparently by termination. Surprisingly, a new 3′-terminal sgRNA was observed from the duplicated 5′TR. A large 3′-terminal sgRNA resulting from the putative promoter activity of the native 5′TR was not observed, possibly because of the down-regulation of a promoter ∼19 kb from the 3′ terminus. However, we were able to observe a sgRNA produced from the native 5′TR of a small defective RNA, which placed the native 5′TR closer to the 3′ terminus, demonstrating sgRNA promoter activity of the native 5′TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5′TR (in the 3′ position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3′- and 5′-terminal sgRNAs. These mutations, when introduced in frame in the native 5′TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3′-terminal sgRNA while having no impact on replication, demonstrating that neither 5′- nor 3′-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5′TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3′ open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3′-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.

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Accumulation of a 5′ proximal subgenomic RNA of Citrus tristeza virus is correlated with encapsidation by the minor coat protein

Virology ◽

10.1016/j.virol.2009.04.009 ◽

2009 ◽

Vol 389 (1-2) ◽

pp. 122-131 ◽

Cited By ~ 11

Author(s):

Siddarame Gowda ◽

Satyanarayana Tatineni ◽

Svetlana Y. Folimonova ◽

Mark E. Hilf ◽

William O. Dawson

Keyword(s):

Coat Protein ◽

Citrus Tristeza Virus ◽

Subgenomic Rna ◽

Tristeza Virus ◽

Minor Coat Protein ◽

Citrus Tristeza

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Erratum to “Accumulation of a 5′ proximal subgenomic RNA of Citrus tristeza virus is correlated with encapsidation by the minor coat protein” [Virology 389 (2009) 122–131]

Virology ◽

10.1016/j.virol.2009.06.033 ◽

2009 ◽

Vol 392 (1) ◽

pp. 149-151

Author(s):

Siddarame Gowda ◽

Satyanarayana Tatineni ◽

Svetlana Y. Folimonova ◽

Mark E. Hilf ◽

William O. Dawson

Keyword(s):

Coat Protein ◽

Citrus Tristeza Virus ◽

Subgenomic Rna ◽

Tristeza Virus ◽

Minor Coat Protein ◽

Citrus Tristeza

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Citrus Tristeza Virus: Molecular Approaches to Cross Protection

10.32747/1994.7570551.bard ◽

1994 ◽

Author(s):

William O. Dawson ◽

Moshe Bar-Joseph ◽

Charles L. Niblett ◽

Ron Gafny ◽

Richard F. Lee ◽

...

Keyword(s):

Citrus Tristeza Virus ◽

Complex Structure ◽

Genetic System ◽

Aphid Transmission ◽

Open Reading Frames ◽

Molecular Approaches ◽

Defective Rnas ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Tristeza

Citrus tristeza virus (CTV) has the largest genomes among RNA viruses of plants. The 19,296-nt CTV genome codes for eleven open reading frames (ORFs) and can produce at least 19 protein products ranging in size from 6 to 401 kDa. The complex biology of CTV results in an unusual composition of CTV-specific RNAs in infected plants which includes multiple defective RNAs and mixed infections. The complex structure of CTV populations poses special problems for diagnosis, strain differentiation, and studies of pathogenesis. A manipulatable genetic system with the full-length cDNA copy of the CTV genome has been created which allows direct studies of various aspects of the CTV biology and pathology. This genetic system is being used to identify determinants of the decline and stem-pitting disease syndromes, as well as determinants responsible for aphid transmission.

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