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 on 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, cytidine monophosphate 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 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, that 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.

A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses

“…For our current study, we characterized the function of a poorly studied BEACH (beige and Chediak–Higashi)- and WD40 repeat-containing protein, WDR81. Interestingly, WDR81 interacts with WDR91 [ 27 , 28 ], which was also identified in our work ( Fig 1B and 1C ) and in another screen for proviral factors of reovirus infection [ 46 ].…”

“…These hits include SLC35A1 and CTSL, whose gene products are required for reovirus infection. SLC35A1 is necessary for the cell surface expression of sialic acid, which serves as a receptor for type 3 reovirus strains, such as T3D [ 46 ]. CTSL encodes the endosomal protease, cathepsin L, which is required for capsid disassembly, a necessary step in reovirus cell entry [ 23 ].…”

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

“…In the CRISPR/Cas-9 screen, four functional pathways defined by Gene Ontology (GO) terms were common to both T1L and T3D, including sialic acid biosynthesis and metabolism ( Fig 1D ). Sialic acid is a reovirus attachment factor, and genes involved in sialic acid biosynthesis and metabolism, including Slc35a1 , are required for T3SA+ replication in BV2 cells [ 37 ]. These data provide confidence that the target genes identified in the CRISPR/Cas-9 screen represent biologically significant candidates.…”

“…Only 28 genes in the CRISPR/Cas9 screens using strains T1L and T3D were identified in the siRNA screen using strain T3SA+, 19 of which are ribosomal genes ( Fig 1B , and 1C). Of the nine non-ribosomal genes, several encode proteins required for reovirus entry, including those involved in sialic acid biosynthesis and metabolism ( Nans and Neu ) [ 37 , 38 ] and viral disassembly ( Ctsl ) [ 22 ].…”

Novel Insights into Selected Disease-Causing Mutations within the SLC35A1 Gene Encoding the CMP-Sialic Acid Transporter

“…Due to its terminal position in the glycan structures, sialic acid, and consequently CST, often mediate attachment and internalization of a wide range of viruses, including polyomaviruses, coronaviruses, some rotaviruses, reoviruses [44,45], influenza virus [46] and others. Modulation of the CST expression has been shown to either prevent viral entry and replication [45][46][47] or facilitate it, as is the case for vesicular stomatitis virus (VSV) and mutant PML-associated JC polyomaviruses [48,49]. Additionally, the CST has been proposed to play a role in mediating a pro-apoptotic response during VSV infections [48].…”