Widespread distribution and evolution of poxviral entry-fusion complex proteins in giant viruses

Abstract

AbstractPoxviruses 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 phylumNucleocytoviricota. The 11 EFC genes are present in almost all pox-viruses, with the two smallest, G3 and O3, absent inEntomopoxvirinaeand basal lineages ofChordopoxvirinae. 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 ofNucleocytoviricota, including metagenome-assembled genomes, but are generally absent in viruses infecting algae or non-amoebozoan heterotrophic protists. The A16/G9/J5 and F9/L1 families co-occur, mostly as single copies, in 93% of the non-Poxviridaegiant viruses that have at least one of them. Distribution and phylogenetic patterns suggest that both families originated in the ancestor ofNucleocytoviricota. In addition to thePoxviridaegenes, homologs from each of the otherNucleo-cytoviricotafamilies 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 ofPoxviridaeand other families. Overall, our study reveals widespread distribution of these EFC protein homologs beyond pox-viruses, implies the existence of a conserved membrane fusion mechanism, and sheds light on host range and ancient evolution ofNucleocytoviricota.ImportanceFusion 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 multi-protein entry-fusion complex (EFC). We report that two major families of the EFC proteins are widely distributed within the virus phylumNucleocytoviricota, which include poxviruses and other 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 ofNucleocytoviricota, was passed down to the major subclades ofNucleocytoviricota, 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 lastNucleocytoviricotacommon ancestor.