Signal peptide and N-glycosylation of N-terminal-CD2v determine the hemadsorption of African swine fever virus.

African swine fever virus (ASFV) is the cause of African swine fever (ASF), a devastating disease that affects domestic pigs and wild boar and is currently responsible for the largest animal epidemic. One of the characteristics of ASFV infection, but absent in naturally attenuated strains, is hemadsorption (HAD), a phenomenon that has been linked to virulence. In this study, we have shown that ASFV HAD depends exclusively on the Nt domain of the ASFV CD2v protein during infection. CD2v is a highly glycosylated protein, and we found that glycosylation is essential for HAD. However, despite the higher degree of CD2v glycosylation, only simultaneous N-glycosylation of two Asp residues on the Nt region is the determinant for HAD. On the contrary, we have demonstrated that the presence of a specific signal peptide sequence on CD2v not only influences the degree of CD2v glycosylation but is also critical for HAD, although not for CD2v localization. Finally, we have shown that the CD2v expression during infection of the non-HAD NH/P68 strain is not sufficient for HAD, despite its glycosylation and cell surface localization. Complementation studies of CD2v from NH/P68 with CD2v signal peptides from HAD+ strains indicate (i) that different signal peptides from different genotypes are able to restore HAD and (ii) the existence of a HAD-inhibitory sequence in NH/P68-CD2v. This study lays the molecular basis for ASFV HAD, which could be key for the study of virulent and pathogenic aspects of the virus, as well as the rational development of new vaccines against ASFV.IMPORTANCEAfrican swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines.