African swine fever virus (ASFV) causes a fatal disease with up to 100% mortality in domestic pigs and wild boar (species Sus scrofa), leading to significant economic losses for swine production. Unlike other eukaryotic viruses, ASFV encodes a histone-like nucleic acid-binding protein, pA104R. The A104R gene is highly conserved and present in all ASFV isolates of different genotypes. Moreover, A104R-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 A104R for viral replication in a permissive wild boar cell line (WSL). In this study, we confirmed that A104R is not essential for in vitro replication of ASFV. Loss of A104R did not detectably affect viral DNA replication or RNA transcription, but led to a moderate reduction of virus titers and plaque sizes. Substitution of A104R by a similar ASFV-like element derived from the genome of an Ornithodoros moubata soft tick was not capable of rescuing the deletion mutant phenotype. In contrast, reintroduction of the authentic A104R 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 of the tick-derived A104R protein (pA104R), and performed mass spectrometric analyses of the pA104R interactome. These experiments revealed besides DNA-dependent co-precipitated proteins, also specific, DNA-independent protein-protein interactions of pA104R with other viral and cellular proteins.