The organization of Physcomitrella patens RAD51 genes is unique among eukaryotic organisms

Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants. To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the RAD51 gene, was isolated from the moss Physcomitrella patens and characterized. Two highly homologous RAD51 genes were found to be present. Duplicated RAD51 genes have been found thus far exclusively in eukaryotes with duplicated genomes. Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of Physcomitrella. Comparison of the protein sequences to Rad51 proteins from other organisms showed that both RAD51 genes originated within the group of plant Rad51 proteins. However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins. In contrast to RAD51 genes from other multicellular eukaryotes, the Physcomitrella genes are not interrupted by introns. Because introns are a common feature of Physcomitrella genes, the lack of introns in the RAD51 genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism. The presence of duplicated intronless RAD51 genes is unique among eukaryotes. Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

<div><p>Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, <em>N</em>-acetylneuraminic acid and <em>N</em>-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.</p> </div

Hemagglutination by σ1 mutant viruses.

<p>Purified virions of the strains shown (10¹¹ particles/well) were serially diluted 1∶2 in PBS in 96-well U-bottom plates. Human erythrocytes were washed several times with PBS, resuspended to a concentration of 1% (vol/vol) in PBS, added to virus-containing wells, and incubated at 4°C for 3 h. Results are expressed as log₂ (HA titer). HA titer is defined as 10¹¹ particles divided by the number of particles/HA unit. One HA unit is the particle number sufficient to produce hemagglutination. *** <i>P</i><0.001, as determined by one-way Anova followed by Bonferroni's multiple comparison test.</p

Data collection and refinement statistics for the T1L σ1-GM3 complex.

<p>r.m.s.d. = root-mean-square deviation.</p>*<p>CC_1/2 = correlation coefficient (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003078#ppat.1003078-Karplus1" target="_blank">[90]</a>).</p>**<p>R_free was calculated with 10% of the data.</p

The effect of neuraminidase treatment on T1L infectivity in L cells and MEFs.

<p>(<b>A</b>) L cells or (<b>B</b>) MEFs were treated with <i>A. ureafaciens</i> neuraminidase for 1 h, followed by adsorption of T1L at an MOI of 10 or 100 PFU/cell, respectively. Cells were washed twice with PBS, and fresh medium was added. After incubation at 37°C for 20 h, cells were fixed, and reovirus antigen was detected by indirect immunofluorescence. Nuclei were stained with DAPI. The percentage of infected cells in three fields of view per well was determined. The results are expressed as the mean percent infected cells per well in triplicate wells for two independent experiments. Error bars represent standard deviations. (<b>A</b>) n.s., (<b>B</b>) ***, <i>P</i><0.0001, as determined by two-tailed Student's <i>t</i> test. (<b>C</b>) L cells or MEFs were stained with anti-JAM-A antibody followed by Alexa-488 labeled secondary antibody to measure cell-surface JAM-A expression. Fluorescence was detected by flow cytometry. Cells were gated on forward and side scatter and the mean fluorescence intensity (MFI) of Alexa-488 was quantified. Results shown are from a representative of three experiments each done with duplicate samples.</p

The effect of soluble glycans on T1L infectivity of MEFs.

<p>(<b>A,B</b>) T1L or (<b>C</b>) T3D (10⁷ PFU/well) were pre-incubated with the GM2 (<b>A,C</b>) or GM3 (<b>B</b>) glycan at the concentrations shown for 1 h prior to adsorption to MEFs at a final MOI of 100 PFU/cell. Cells were washed twice with PBS, and fresh medium was added. After incubation at 37°C for 20 h, cells were fixed and reovirus antigen was detected by indirect immunofluorescence. Nuclei were quantified by DAPI staining. The results are expressed as the mean percent infected cells per field in triplicate wells for two independent experiments. Error bars represent standard deviations. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.0001, as determined by two-tailed Student's <i>t</i> test.</p