Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence

Saunders, Keith; Richardson, Jeremy; Lawson, David M.; Lomonossoff, George P.

doi:10.3390/v12111235

“…The need for a nucleic acid scaffold for the efficient production of VLPs of helical viruses parallels the situation found with the ssDNA-containing geminiviruses [ 12 , 31 ]. As with the helical viruses reported here, expression of the coat protein alone does not result in the production of any particles and the presence of replicating ssDNA is essential.…”

Section: Discussionmentioning

confidence: 99%

“…As with the helical viruses reported here, expression of the coat protein alone does not result in the production of any particles and the presence of replicating ssDNA is essential. Furthermore, the precise morphology of the particles is governed by the length of the replicating DNA [ 12 , 31 ]. Though we have not investigated this in the current study, it is probable that the length of the helical VLPs could also be controlled by varying the length of the replicating RNA.…”

Section: Discussionmentioning

confidence: 99%

“…However, in vitro assembly is only feasible on a small scale with a virus, such as TMV, that has been extensively studied. While expression of the coat protein (CP) and self-assembly into VLPs has been very effective with isometric plant viruses [ 5 , 9 , 10 , 11 , 12 ], expression of the coat protein alone in the case of helical viruses generally results in either low yields, very heterogeneous material, or both. For example, the non-replicating pEAQ- HT system [ 13 ] has been used for the production of turnip mosaic virus VLPs by expression of coat protein alone [ 14 ], and tobacco mosaic virus VLPs by co-expression of coat protein with RNA bearing a TMV origin of assembly (OAS; [ 15 ]); however only relatively low yields were reported.…”

Section: Introductionmentioning

confidence: 99%

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A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

Thuenemann

¹

,

Byrne

²

,

Peyret

³

et al. 2021

Viruses

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The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

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“…We extended these observations by showing that SEGS-2 is packaged as ssDNA in Arabidopsis. At 1.2 Kb, SEGS-2 is similar in size to other begomovirus satellites, which are also encapsidated into virions as ssDNA (Hesketh et al, 2018; Saunders et al, 2020). SEGS-2 also occurs as a dsDNA in Arabidopsis leaves, characteristic of rolling circle and/or recombination dependent replication (Hanley-Bowdoin et al, 2013; Jeske, Lütgemeier, and Preiss, 2001) (Fig.…”

Section: Discussionmentioning

confidence: 98%

A New Type of Satellite associated with Cassava Mosaic Begomoviruses

Aimone

¹

,

Leon

²

,

Dallas

³

et al. 2021

Preprint

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Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated as SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is co-inoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus resistant Arabidopsis accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in Arabidopsis. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of a SEGS-2 encoded protein that may also be encoded in the cassava genome.

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“…We extended these observations by showing that SEGS-2 is packaged as ssDNA in A. thaliana . At 1.2 kb, SEGS-2 is similar in size to other begomovirus satellites, which are also encapsidated into virions as ssDNA ( 46 , 47 ). SEGS-2 also occurs as a dsDNA in A. thaliana leaves, characteristic of rolling-circle and/or recombination-dependent replication ( 13 , 48 ) ( Fig.…”

Section: Discussionmentioning

confidence: 99%

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses

Aimone

¹

,

Leon

²

,

Dallas

³

et al. 2021

J Virol

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Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated as SEGS-2 ( s equence e nhancing g eminivirus s ymptoms). We report here that when SEGS-2 is co-inoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana , viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana . Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of a SEGS-2 encoded protein that may also be encoded in the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world’s cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

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Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence

Cited by 11 publications

References 56 publications

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

A New Type of Satellite associated with Cassava Mosaic Begomoviruses

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses

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