Tim L. Sit

Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus, Revealing a Potential Mechanism for RNA Release

ABSTRACT The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 ± 30 Ca2+ ions bound to the capsid and 420 ± 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-Å-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.

A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

The Red clover necrotic mosaic virus RNA-2 encoded movement protein is a second suppressor of RNA silencing

The replication complex of Red clover necrotic mosaic virus (RCNMV) has been shown to possess silencing suppression activity. Here a newly developed viral-based assay for the identification of silencing suppression activity was used to provide evidence for a second, mechanistically distinct method of silencing suppression provided for by the RCNMV movement protein (MP). This new assay relies on Turnip crinkle virus with its capsid protein replaced with green fluorescent protein to act as a reporter (TCV-sGFP). In the presence of a protein with silencing suppression activity TCV-sGFP readily moves from cell-to-cell, but in the absence of such a protein TCV-sGFP is confined to small foci of infection. This TCV-sGFP assay was used to identify MP as a suppressor of RNA silencing, to delimit essential amino acids for this activity and uncouple silencing and movement functions.

Gene expression throughout a vertebrate's embryogenesis

BackgroundDescribing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage.ResultsPatterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes.ConclusionsThese data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.

The Red clover necrotic mosaic virus origin of assembly is delimited to the RNA-2 trans-activator.

The bipartite RNA genome of Red clover necrotic mosaic virus (RCNMV) is encapsidated into icosahedral virions that exist as two populations: i) virions that co-package both genomic RNAs and ii) virions packaging multiple copies of RNA-2. To elucidate the packaging mechanism, we sought to identify the RCNMV origin of assembly sequence (OAS). RCNMV RNA-1 cannot package in the absence of RNA-2 suggesting that it does not contain an independent packaging signal. A 209 nt RNA-2 element expressed from the Tomato bushy stunt virus CP subgenomic promoter is co-assembled with genomic RNA-1 into virions. Deletion mutagenesis delimited the previously characterized 34 nt trans-activator (TA) as the minimal RCNMV OAS. From this study we hypothesize that RNA-1 must be base-paired with RNA-2 at the TA to initiate co-packaging. The addition of viral assembly illustrates the critical importance of the multifunctional TA element as a key regulatory switch in the RCNMV life cycle.

A Single Amino Acid Mutation in the Carnation Ringspot Virus Capsid Protein Allows Virion Formation but Prevents Systemic Infection

ABSTRACT A Carnation ringspot virus (CRSV) variant (1.26) was identified that accumulates virions but is incapable of forming a systemic infection. The 1.26 capsid protein gene possesses a Ser→Pro mutation at amino acid 282. Conversion of 1.26 amino acid 282 to Ser restored systemic infection, while the reciprocal mutation in wild-type CRSV abolished systemic infection. Similar mutations introduced into the related Red clover necrotic mosaic virus capsid protein gene failed to induce the packaging but nonsystemic movement phenotype. These results provide additional support for the theory that virion formation is necessary but not sufficient for systemic movement with the dianthoviruses.

Development of abamectin loaded plant virus nanoparticles for efficacious plant parasitic nematode control.

Plant parasitic nematodes are one of the worlds major agricultural pests, causing in excess of

Loading and Release Mechanism of Red Clover Necrotic Mosaic Virus Derived Plant Viral Nanoparticles for Drug Delivery of Doxorubicin

157 billion in worldwide crop damage annually. Abamectin (Abm) is a biological pesticide with a strong activity against a wide variety of plant parasitic nematodes. However, Abms poor mobility in the soil compromises its nematicide performance because of the limited zone of protection surrounding the growing root system of the plant. In this study, we manipulated Abms soil physical chemistry by encapsulating Abm within the Red clover necrotic mosaic virus (RCNMV) to produce a plant virus nanoparticle (PVN) delivery system for Abm. The transmission electron microscopic and dynamic light scattering characterization of Abm-loaded PVN (PVN(Abm)) indicated the resultant viral capsid integrity and morphology comparable to native RCNMV. In addition, the PVN(Abm) significantly increased Abms soil mobility while enabling a controlled release strategy for Abms bioavailability to nematodes. As a result, PVN(Abm) enlarged the zone of protection from Meloidogyne hapla root knot nematodes in the soil as compared to treating with free Abm molecules. Tomato seedlings treated with PVN(Abm) had healthier root growth and a reduction in root galling demonstrating the success of this delivery system for the increased efficacy of Abm to control nematode damage in crops.

Genomic and physiological responses to strong selective pressure during late organogenesis: few gene expression changes found despite striking morphological differences

Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMVs structure and unique response to divalent cation depletion and re-addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface-binding or internal capsid-encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface-Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH-responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox-loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsids pH- and ion- responsive functions in a given environment.

The Red clover necrotic mosaic virus capsid protein N-terminal lysine-rich motif is a determinant of symptomatology and virion accumulation.

BackgroundAdaptations to a new environment, such as a polluted one, often involve large modifications of the existing phenotypes. Changes in gene expression and regulation during critical developmental stages may explain these phenotypic changes. Embryos from a population of the teleost fish, Fundulus heteroclitus, inhabiting a clean estuary do not survive when exposed to sediment extract from a site highly contaminated with polycyclic aromatic hydrocarbons (PAHs) while embryos derived from a population inhabiting a PAH polluted estuary are remarkably resistant to the polluted sediment extract. We exposed embryos from these two populations to surrogate model PAHs and analyzed changes in gene expression, morphology, and cardiac physiology in order to better understand sensitivity and adaptive resistance mechanisms mediating PAH exposure during development.ResultsThe synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (β-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (α-naphthoflavone), caused significant developmental delays, impaired cardiac function, severe morphological alterations and failure to hatch, leading to the deaths of reference embryos; resistant embryos were mostly unaffected. Unexpectedly, patterns of gene expression among normal and moderately deformed embryos were similar, and only severely deformed embryos showed a contrasting pattern of gene expression. Given the drastic morphological differences between reference and resistant embryos, a surprisingly low percentage of genes, 2.24% of 6,754 analyzed, show statistically significant differences in transcript levels during late organogenesis between the two embryo populations.ConclusionsOur study demonstrates important contrasts in responses between reference and resistant natural embryo populations to synergistic effects of surrogate model PAHs that may be important in adaptive mechanisms mediating PAH effects during fish embryo development. These results suggest that statistically significant changes in gene expression of relatively few genes contribute to the phenotypic changes and large morphological differences exhibited by reference and resistant populations upon exposure to PAH pollutants. By correlating cardiac physiology and morphology with changes in gene expression patterns of reference and resistant embryos, we provide additional evidence for acquired resistance among embryos whose parents live at heavily contaminated sites.