Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion

Abstract

ABSTRACTAfrican swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. The virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. As several other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. Endosomal maturation entails luminal acidification and the lowering of pH acting on the multi-layered virion structure dissolves the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Egress from endosome is related to cholesterol efflux, but it remains an intriguing process albeit essential for infection, specifically for the viral nucleic acid exit to the cytoplasm for replication. ASFV proteins E248R and E199L, with structural homology to the VACV proteins of the fusion complex, seem to have similar functions in ASFV. A direct interaction between these ASFV proteins with the cholesterol transporter protein NPC1 (Niemann-Pick C type 1) was observed, which was also shared by the E248R homologous protein L1R of VACV. Binding occurs between the transmembrane domain of E248R with the loop C of NPC1 at the same domain than EBOV binding site. These interactions suggest that these ASFV proteins are crucial for membrane fusion. CRISPR NPC1 KO Vero cells lacking NPC1 protein that were resistant to EBOV, reduced ASFV infection levels significantly. Reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by lesser viral factories of smaller size and lacking the typical cohesive morphology between endosomes and viral proteins. We observed a compensatory effect in NPC1 KO cells, elevating NPC2 levels while silencing NPC2 in Vero cells with shRNA, also reduced ASFV infection. Our findings pave the way to understand the role of these proteins at the membrane viral fusion step for several viruses.Author SummaryAfrican swine fever virus (ASFV) causes a deadly disease of pigs and wild boars that was endemic in Africa but have extended over the last years to Europe, Asia and Oceania with high socioeconomic impact. ASFV enters the cell by endocytosis and has adapted to the endosomal conditions to acquire infectivity. Viral infectivity is dependent on cholesterol traffic at the endosomes, especially at the fusion step. Fusion of the internal viral membrane with the endosomal membrane is required for the exit of the viral DNA to the cytoplasm to start replication. ASF virion internal membrane proteins E248R and E199L were found to bind the Niemann Pick C1 (NPC1) receptor at the endosome. These proteins are highly conserved among ASFV isolates and resemble proteins of the VACV entry/fusion complex. The function of NPC1 is to regulate the efflux of dietary cholesterol efflux from the endosome to the endoplasmic reticulum, which appears to be necessary for viral fusion. NPC1 knockout cells by CRISPR reduced infection affecting infectivity and early replication. Also, removing the associated endosomal protein NPC2, further declined infectivity. These results show the relevance of NPC1 receptor in the viral infection actually shared by unrelated important viral families.