Functional Analyses of the Histone-like A104R Protein of African Swine Fever Virus and of a Homologous Pseudogene Product Found in Soft Tick Genomes

African swine fever virus (ASFV) causes a fatal disease in domestic pigs and wild boars (<i>Sus scrofa</i>), leading to nearly 100% mortality during acute infection and significant economic losses in swine production. Unlike other eukaryotic viruses, ASFV encodes a histone-like nucleic acid-binding protein, pA104R, which is highly conserved and present in all described ASFV isolates of different genotypes. Moreover, <i>A104R</i>-like sequences have been identified in the genomes of soft ticks, which can replicate and transmit ASFV. Using a virulent genotype IX field isolate from Kenya, we analyzed the importance of <i>A104R</i> for viral replication in a permissive wild boar cell line (WSL). In this study, we confirmed that <i>A104R</i> is not essential for in vitro replication of ASFV. Loss of <i>A104R</i> did not detectably affect viral DNA replication or RNA transcription but led to a moderate reduction in virus titers and plaque sizes. Substitution of <i>A104R</i> with a similar ASFV-like element derived from the genome of an <i>Ornithodoros moubata</i> soft tick was not capable of rescuing the deletion mutant phenotype. In contrast, reintroduction of the authentic <i>A104R</i> open reading frame (ORF) into the deletion mutant fully restored wild-type virus growth properties. In accompanying studies, we verified the DNA-binding activities of the ASFV- and tick-derived A104R proteins and performed mass spectrometric analyses of the pA104R interactome. These experiments revealed, besides DNA-dependent co-precipitated proteins, specific DNA-independent protein-protein interactions of pA104R with other viral and cellular proteins.