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1.  Deciphering the Mechanism of Defective Interfering RNA (DI RNA) Biogenesis Reveals That a Viral Protein and the DI RNA Act Antagonistically in Virus Infection 
Journal of Virology  2013;87(11):6091-6103.
Potato mop-top virus (PMTV) produces a defective RNA (D RNA) encompassing the 5′-terminal 479 nucleotides (nt) and 3′-terminal 372 nt of RNA-TGB (where TGB is triple gene block). The mechanism that controls D RNA biogenesis and the role of D RNA in virus accumulation was investigated by introducing deletions, insertions, and point mutations into the sequences of the open reading frames (ORFs) of TGB1 and the 8-kilodalton (8K) protein that were identified as required for efficient production of the D RNA. Transient expression of RNA-TGB in the absence of RNA-Rep (which encodes the replicase) did not result in accumulation of D RNA, indicating that its production is dependent on PMTV replication. The D RNA could be eliminated by disrupting a predicted minus-strand stem-loop structure comprising complementary sequences of the 5′ TGB1 ORF and the 3′ 8K ORF, suggesting intramolecular template switching during positive-strand synthesis as a mechanism for the D RNA biogenesis. Virus accumulation was reduced when the 8K ORF was disrupted but D RNA was produced. Conversely, the virus accumulated at higher titers when the 8K ORF was intact and D RNA production was blocked. These data demonstrate that the D RNA interferes with virus infection and therefore should be referred to as a defective interfering RNA (DI RNA). The 8K protein was shown to be a weak silencing suppressor. This study provides an example of the interplay between a pathogen and its molecular parasite where virus accumulation was differentially regulated by the 8K protein and DI RNA, indicating that they play antagonistic roles and suggesting a mechanism by which the virus can attenuate replication, decreasing viral load and thereby enhancing its efficiency as a parasite.
doi:10.1128/JVI.03322-12
PMCID: PMC3648117  PMID: 23514891
2.  Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform 
PLoS ONE  2013;8(11):e79235.
A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.
doi:10.1371/journal.pone.0079235
PMCID: PMC3828288  PMID: 24244456
3.  Novel Bacteriophages Containing a Genome of Another Bacteriophage within Their Genomes 
PLoS ONE  2012;7(7):e40683.
A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3′ protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a ‘fast track’ route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.
doi:10.1371/journal.pone.0040683
PMCID: PMC3398947  PMID: 22815791
4.  The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids 
The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1–2 μm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.
doi:10.3389/fpls.2012.00290
PMCID: PMC3529358  PMID: 23269927
PMTV; TGB2; viral RNA; replication; chloroplasts; inclusions
5.  Unusual Features of Pomoviral RNA Movement 
Potato mop-top pomovirus (PMTV) is one of a few viruses that can move systemically in plants in the absence of the capsid protein (CP). Pomoviruses encode the triple gene block genetic module of movement proteins (TGB 1, 2, and 3) and recent research suggests that PMTV RNA is transported either as ribonucleoprotein (RNP) complexes containing TGB1 or encapsidated in virions containing TGB1. Furthermore, there are different requirements for local or systemic (long-distance) movement. Research suggests that nucleolar passage of TGB1 may be important for the long-distance movement of both RNP and virions. Moreover, and uniquely, the long-distance movement of the CP-encoding RNA requires expression of both major and minor CP subunits and is inhibited when only the major CP sub unit is expressed. This paper reviews pomovirus research and presents a current model for RNA movement.
doi:10.3389/fmicb.2011.00259
PMCID: PMC3244614  PMID: 22203822
Pomovirus; PMTV; BVQ; BSBV; nucleus; nucleolus; microtubules; TGB
6.  In Situ Spatial Organization of Potato Virus A Coat Protein Subunits as Assessed by Tritium Bombardment 
Journal of Virology  2001;75(20):9696-9702.
Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing α-helices and β-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two β-strands, (ii) a C-terminal region including two α-helices, as well as three β-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four α-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.
doi:10.1128/JVI.75.20.9696-9702.2001
PMCID: PMC114541  PMID: 11559802

Results 1-6 (6)