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A CRISPR screen using BV2 cells identifies sialic acid synthesis genes as required for reovirus attachment and infection

Urbanek, Kelly (2019) A CRISPR screen using BV2 cells identifies sialic acid synthesis genes as required for reovirus attachment and infection. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Engagement of cell-surface receptors by viruses is a critical determinant of tropism and disease. The reovirus attachment protein, σ1, first binds sialylated 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 replication and subsequent cell killing, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus-induced 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 T3SA+, a strain known to engage sialic acid, and no change in the low-level binding of T3SA-, a strain that does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in CRISPR-modified cells. Following exogenous expression of Cmas and Slc35a1 into the respective null cells, sialic acid expression was restored. These results demonstrate that Cmas and Slc35a1, which are required for cell-surface expression of sialic acid, enhance reovirus attachment. Moreover, these findings provide additional evidence that sialic acid, which is expressed on most cells, serves as an attachment factor for reovirus. While reovirus is currently not a major public health concern, reoviruses are a highly tractable experimental model for the study of viral pathogenesis. These findings shed light on general principles of virus-receptor interactions which are important determinants of dissemination and tropism of many viruses affecting public health today.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Urbanek, Kellyklm179@pitt.eduklm179
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberDemirci, F. Yesimfyd1@pitt.edu
Committee MemberHartman, Amyhartman2@pitt.edu
Committee MemberWilliams, Johnjvw@chp.edu
Thesis AdvisorDermody, Terenceterence.dermody@chp.edu
Date: 26 July 2019
Date Type: Publication
Defense Date: 8 May 2019
Approval Date: 26 July 2019
Submission Date: 3 June 2019
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 68
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Human Genetics
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Cmas; Slc35a1; Sialic acid; Reovirus
Date Deposited: 26 Jul 2019 20:33
Last Modified: 26 Jul 2019 20:33
URI: http://d-scholarship.pitt.edu/id/eprint/36878

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