Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection

Abstract

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA⁺ and T3SA^-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA⁺, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA^-, which does not engage sialic acid. Furthermore, infectivity of T3SA⁺ was diminished to levels similar to those of T3SA^- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA⁺ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

Keywords: cytidine monophosphate N-acetylneuraminic acid synthetase; microglia; reovirus; sialic acid; solute carrier family 35 member A1; viral attachment.

FIG 1

Whole-genome CRISPR screen identifies sialic acid (SA) synthesis genes as required for reovirus-induced death of BV2 cells. (A) The top 10 candidates from CRISPR screens using reovirus strains T3SA⁺ and T3SA⁻ are ranked by their STARS scores. The heat map indicates STARS values. Genes encoding proteins involved in SA synthesis are indicated by blue shading. (B) Comparison of STARS scores from the candidates of the BV2 CRISPR screen between the T3SA⁺ (x axis) and T3SA⁻ (y axis) conditions. Genes that did not meet the criteria to receive a STARS score are assigned a value of 0. Genes linked to SA metabolism are colored in red.

FIG 2

SA expression is diminished following CRISPR-mediated gene disruption and restored by stable complementation of Cmas and Slc35a1 genes. Cells were incubated with fluorescein-labeled WGA to assess cell surface expression of SA. WGA binding to cells was detected by flow cytometry. (A) Representative flow cytometric profiles of fluoresceinated WGA binding to WT, CRISPR-ablated, and complemented cells. (B) The mean fluorescence intensity (MFI) of WGA binding was quantified. The data represent results from three independent experiments each with duplicate samples. Error bars indicate standard error of the mean (SEM). *, P < 0.05; **, P < 0.01; ns, not significant, as determined by Welch’s analysis of variance (ANOVA) with Dunnett’s multiple-comparison test.

FIG 3

BV2 cells lacking Cmas or Slc35a1 expression are protected from reovirus-induced cell death, while complemented cells are not. The cell lines shown were adsorbed with (A) T3SA⁻ or (B) T3SA⁺ at an MOI of 100 PFU/cell, and cell viability was quantified using a PrestoBlue assay at the intervals shown. Data are normalized to a mock-infected condition (not shown) and the 0 h average for each respective cell line. Data are representative of results from nine technical replicates from three independent experiments. Error bars indicate SEM.

FIG 4

Expression of Cmas and Slc35a1 is required for reovirus infection of BV2 cells. The cell lines shown were adsorbed with T3SA⁺ or T3SA⁻ at an MOI of 100 PFU/cell. The percentage of infected cells was determined by enumeration of reovirus-positive cells at 24 h postadsorption from immunofluorescence images. The data are representative of results from nine technical replicates from three independent experiments. Error bars indicate SEM. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001, as determined by Welch’s ANOVA with Dunnett’s multiple-comparison test.

FIG 5

Expression of Cmas and Slc35a1 confers reovirus binding to BV2 cells. The cell lines shown were adsorbed with 10⁵ particles/cell of Alexa Fluor 647-labeled T3SA⁺ (A) or T3SA⁻ (B). Virus binding was quantified using flow cytometry. Representative flow cytometric profiles are shown. (C) The MFI of Alexa Fluor 647 was quantified. The data are representative of results from six technical replicates from three independent experiments. Error bars represent SEM. **, P < 0.01; ***, P < 0.001; ns, not significant, as determined by Welch’s ANOVA with Dunnett’s multiple-comparison test.