Giant viruses have garnered significant attention in virological research due to their large genomes and unique gene content. As a member of the giant virus family, Marseillevirus contains several genes related to protein translation in its genome. This study aims to investigate the function of MarSH_314 of Marseillevirus Shanghai strain (Mar-SH2016), which was annotated to be the eukaryotic translation initiation factor eIF2/eIF5. We constructed a strain of Acanthamoeba castellanii overexpressing MarSH_314, and identified the interacting proteins of MarSH_314 through co-immunoprecipitation (Co-IP) and mass spectrometry analysis. Mass spectrometry and following biochemical results suggested a potential interaction between MarSH_314 and the host transcriptional repressor Tup1. When interfering with the expression of Tup1 using siRNA, viral replication was significantly inhibited. The result did not support MarSH_314 as a translation factor, instead, it suggested that MarSH_314 has an important role in virus replication through interacting with Tup1.