Abstract Genome-wide genetic screens are powerful tools to identify genes that act as host factors of viruses. We have applied this technique to the 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 β 2 -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 β 2 -microglobulin as a relevant factor in the Vaccinia virus entry process. Author summary Orthopoxviruses, a genus belonging to the family Poxviridae , include human pathogens like Variola virus, the causative agent of the now eradicated Smallpox, and Monkeypox virus that cause human outbreaks of zoonotic origin. Being the prototype Poxvirus, Vaccinia virus has been extensively used as the ideal model to study infection. For Poxviruses, both fluid phase endocytosis and direct fusion at the plasma membrane have been described as modes of entry. To date, only a few cellular factors have been identified in the vaccinia virus entry pathway. In this study, we report that blind genome-wide genetic screens allowed us to identify several cellular factors involved in Vaccinia Virus infection, of which many could be related to known factors in virus entry. In addition, we found that β 2 -microglobulin constitute a novel player for Poxvirus entry not related to previously described cellular pathways involved in the entry process. These findings add new information to the complex picture of Poxvirus entry and open the door to the discovery of new entry mechanisms used by Poxviruses.

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