Abstract
Viruses of the phylum Nucleocytoviricota, or nucleo-cytoplasmic large DNA viruses (NCLDVs), undergo a cytoplasmic or nucleo-cytoplasmic cycle, the latter of which involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting NCLDVs. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate its infection strategy, we performed a time course RNA sequencing (RNA-seq) experiment. All viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and often (45%) encoded putative nucleus-localized proteins. These results suggest massive reshaping of the host nuclear environment by viral proteins at an early stage of infection. Genes in other expression clusters (clusters 2 to 5) were assigned to various functional categories. The virally encoded core histone genes were in cluster 3, whereas the viral linker histone H1 gene was in cluster 1, suggesting they have distinct roles during the course of the virus infection. The transcriptional profile of the host Acanthamoeba castellanii genes was greatly altered postinfection. Several encystment-related host genes showed increased representation levels at 48 h postinfection, which is consistent with the previously reported amoeba encystment upon medusavirus infection. IMPORTANCE Medusavirus is an amoeba-infecting giant virus that was isolated from a hot spring in Japan. It belongs to the proposed family "Medusaviridae" in the phylum Nucleocytoviricota. Unlike other amoeba-infecting giant viruses, medusavirus initiates its DNA replication in the host nucleus without disrupting the nuclear membrane. Our RNA sequencing (RNA-seq) analysis of its infection course uncovered ordered viral gene expression profiles. We identified temporal expression clusters of viral genes and associated putative promoter motifs. The subcellular localization prediction showed a clear spatiotemporal correlation between gene expression timing and localization of the encoded proteins. Notably, the immediate early expression cluster was enriched in genes targeting the nucleus, suggesting the priority of remodeling the host intranuclear environment during infection. The transcriptional profile of amoeba genes was greatly altered postinfection.