Widespread Distribution and Evolution of Poxviral Entry-Fusion Complex Proteins in Giant Viruses.

Poxviruses are known to encode a set of proteins that form an entry-fusion complex (EFC) to mediate virus entry. However, the diversity, evolution, and origin of these EFC proteins remain poorly understood. Here, we identify the EFC protein homologs in poxviruses and other giant viruses of the phylum . The 11 EFC genes are present in almost all poxviruses, with the two smallest, G3 and O3, being absent in and basal lineages of . Five of the EFC genes are further grouped into two families, A16/G9/J5 and F9/L1, which are widely distributed across other major lineages of , including metagenome-assembled genomes, but are generally absent in viruses infecting algae or nonamoebozoan heterotrophic protists. The A16/G9/J5 and F9/L1 families cooccur, mostly as single copies, in 93% of the non- giant viruses that have at least one of them. Distribution and phylogenetic patterns suggest that both families originated in the ancestor of . In addition to the genes, homologs from each of the other families are largely clustered together, suggesting their ancient presence and vertical inheritance. Despite deep sequence divergences, we observed noticeable conservation of cysteine residues and predicted structures between EFC proteins of and other families. Overall, our study reveals widespread distribution of these EFC protein homologs beyond poxviruses, implies the existence of a conserved membrane fusion mechanism, and sheds light on host range and ancient evolution of . Fusion between virus and host membranes is critical for viruses to release genetic materials and to initiate infection. Whereas most viruses use a single protein for membrane fusion, poxviruses employ a multiprotein entry-fusion complex (EFC). We report that two major families of the EFC proteins are widely distributed within the virus phylum , which includes poxviruses and other double-stranded (dsDNA) giant viruses that infect animals, amoebozoans, algae, and various microbial eukaryotes. Each of these two protein families is structurally conserved, traces its origin to the root of , was passed down to the major subclades of , and is retained in most giant viruses known to infect animals and amoebozoans. The EFC proteins therefore represent a potential mechanism for virus entry in diverse giant viruses. We hypothesize that they may have facilitated the infection of an animal/amoebozoan-like host by the last common ancestor.