Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth

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

Crystal structure of T1L σ1 in complex with the GM2 glycan.

<p>Ribbon tracing of the complex viewed from the side (<b>A</b>) with a close-up of the carbohydrate-binding site (<b>B</b>) and top-view of the complex (<b>C</b>). The three T1L σ1 monomers are depicted in blue, red, and yellow. β-spiral repeats 1, 2, and 3 and β-strands A-H are labeled. The GM2 oligosaccharide is shown in stick representation, with carbons, oxygens, and nitrogens colored yellow, red, and blue, respectively. An unbiased F_o-F_c map of the carbohydrate is shown at a contour level of 3 σ for 2.0 Å around the GM2 glycan (see Materials and Methods section). (<b>D</b>) Schematic representation of the σ1 domain organization. Binding sites in T1L and T3D σ1 for JAM-A and carbohydrate are depicted in green and pink, respectively.</p

T1L reovirus uses GM2 as a coreceptor.

<p>(<b>A</b>) Glycan microarray analysis of recombinant T1L σ1_short using 21 lipid-linked oligosaccharide probes. Each oligosaccharide probe was arrayed at four levels (as indicated) in duplicate. Numerical scores of the binding signals are means of duplicate spots (with error bars). The complete list of probes and their sequences are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003078#ppat.1003078.s006" target="_blank">Table S2</a>. (<b>B</b>) Diagrams of “a series” gangliosides GM3, GM2, GM1, and GD1a present in the glycan array. Ceramide, glucose (Glc), galactose (Gal), <i>N</i>-5-acetyl neuraminic acid (Neu5Ac), and <i>N</i>-acetylgalactosamine (GalNAc) moieties are indicated. (<b>C</b>) STD NMR spectroscopy demonstrates that T1L σ1 binds to the GM2 glycan in solution. Upper spectrum: ¹H spectrum of the GM2 oligosaccharide alone; middle: STD spectrum of T1L σ1 and the GM2 glycan; and lower spectrum: STD spectrum of the GM2 glycan alone as a control for direct excitation of the ligand. The protons are labeled and color-coded according to the sugar moieties within the GM2 oligosaccharide. The large peak just below 3.8 ppm unites the Neu5Ac H4 and H8 and the GalNAc H6 resonances.</p

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