Identification of β2 microglobulin, the product of B2M gene, as a Host Factor for Vaccinia Virus Infection by Genome-Wide CRISPR genetic screens

Abstract

Genome-wide genetic screens are powerful tools to identify genes that act as host factors of viruses. We have applied this technique to analyze the infection of HeLa cells by Vaccinia virus, in an attempt to find genes necessary for infection. Infection of cell populations harboring single gene inactivations resulted in no surviving cells, suggesting that no single gene knock-out was able to provide complete resistance to Vaccinia virus and thus allow cells to survive infection. In the absence of an absolute infection blockage, we explored if some gene inactivations could provide partial protection leading to a reduced probability of infection. Multiple experiments using modified screening procedures involving replication restricted viruses led to the identification of multiple genes whose inactivation potentially increase resistance to infection and therefore cell survival. As expected, significant gene hits were related to proteins known to act in virus entry, such as ITGB1 and AXL as well as genes belonging to their downstream related pathways. Additionally, we consistently found β2-microglobulin, encoded by the B2M gene, among the screening top hits, a novel finding that was further explored. Inactivation of B2M resulted in 54% and 91% reduced VV infection efficiency in HeLa and HAP1 cell lines respectively. In the absence of B2M, while virus binding to the cells was unaffected, virus internalization and early gene expression were significantly diminished. These results point to β2-microglobulin as a relevant factor in the Vaccinia virus entry process.

Spatio-temporal analysis of Vaccinia virus infection and host response dynamics using single-cell transcriptomics and proteomics

“…Roquin-1 is also known to modulate miRNA stability leading to mRNA silencing by interacting with Dicer and Ago proteins 55,56 . Furthermore, our recent findings, and supported by other studies, showed that RC3H1 has a proviral role in VV infection [57][58][59] . Taken together, our observations suggest that VV manipulates the host's anti-inflammatory mechanisms precisely and add polyA tail-mediated mRNA destabilization to the list of known VV-mediated mRNA depletion strategies.…”

“…Our analyses show that for most host factors, the up or downregulation at both mRNA and protein levels align. Notably, we observed the relative upregulation (transcript and protein) for Roquin-1 (RC3H1), a pro-VV factor previously identified in our genetic screens 58 . RC3H1 is an E3 ubiquitin ligase with a RING domain and an RNA-binding CCCH zinc finger, involved in immune homeostasis and in promoting immune-relevant mRNAs downregulation through polyA removal [51][52][53][54]93,94 .…”

MaGplotR: a software for the analysis and visualization of multiple MaGeCK screen datasets through aggregation

“…This tool has been tested using multiple datasets to offer robust and wide applicability [16][17][18][19][20]. Of note, this tool can be used to detect hits that were previously unnoticed, by aggregating common hits from different experiments, as is the case of GATA6 proviral gene for SARS-CoV-2, which we identified as the 4 th hit using rearranged Biering et al datasets [19], and has been confirmed by Israeli et al [21].…”