Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus.

Crimean Congo hemorrhagic fever virus (CCHFV) causes fatal tick-borne disease in humans and is a priority pathogen of the World Health Organization. No licensed vaccines or specific antiviral drugs are available. To understand the cell entry of CCHFV and identify potential antiviral targets to combat the disease, here, we perform the CRISPR knockout screen in wild-type cells, followed by a complementary CRISPR activation screen in cells deficient in common attachment factors (heparan sulfate, AXL, TIM-1). We identify the soluble milk fat globule-EGF factor 8 protein (MFGE8), also known as lactadherin, as a proviral factor for CCHFV infection. Overexpression of MFGE8 enhances the pseudotyped, tecVLP, and authentic CCHFV infection, while knockout decreases the infection. MFGE8 is found to promote the virus binding and internalization. Expression of MFGE8 with D48E mutation of the RGD motif and the use of pharmacological inhibitor and gene-editing suggests that MFGE8 mediates virus entry through integrin receptors on the cell surface. Further study demonstrates that soluble MFGE8 protein acts as a bridge to support the entry by binding to not only the reported phosphatidylserine (PtdSer) but also Gc protein on viral envelope and to integrins on cells. The finding of MFGE8 that can bind directly to Gc protein and the entry mode of CCHFV that employs a soluble protein may expand the tissue tropism and increase the pathogenicity of CCHFV. Our study also provides new insight into the underlying mechanisms of cell entry and development of countermeasures for CCHFV. IMPORTANCE: CCHFV causes severe hemorrhagic fever outbreaks, with a mortality rate of up to 40%. Countries generally list CCHFV as one of the pathogens that requires the highest biosafety level 4 (BSL-4) of containment, which hinders the study of its cell biology and pathogenesis. LDLR was recently identified as a receptor for CCHFV, but other receptors or co-factors remain to be explored. We perform genome-wide CRISPR screens using a safe replication-competent CCHFV pseudovirus and identify a secretory MFGE8 protein that functions as an entry mediator by binding to both the Gc protein and PtdSer on the viral envelope and to the integrins on the cells. Cell entry mediated by a soluble protein may greatly expand the tissue tropism, and the strategies developed to disturb the interaction of MFGE8 with virions or with integrins may help to mitigate the fatal disease induced by CCHFV.