Sputnik virophage disrupts the transcriptional regulation of its host giant virus

Sputnik virophages are small double-stranded DNA (dsDNA) viruses that replicate only inside host amoebae infected with giant dsDNA viruses, mimiviruses. Sputnik infection affects mimivirus replication, but their molecular interaction remains poorly understood. Here, we performed a time-course transcriptome analysis of <i>Acanthamoeba castellanii</i> cells infected with Acanthamoeba polyphaga mimivirus (APMV; hereafter referred to as Sputnik^- cells) and those infected with both APMV and Sputnik 3 virophage (Sputnik⁺ cells). The gene expression patterns of the amoeba were similar between these two conditions, whereas the expression of APMV genes was drastically affected by Sputnik, depending on the timing of their expression. Early-expressed APMV genes showed similar expression patterns in both conditions at the early stage of infection. However, at later stages, their expression levels remained higher in Sputnik⁺ cells than in Sputnik^- cells, suggesting a prolongation of early gene expression by Sputnik. Late-expressed APMV genes showed lower expression at earlier stages in Sputnik⁺ cells, but their expression levels reached or exceeded those in Sputnik^- cells at later stages, indicating a delay in gene expression. Overall, our results demonstrated that Sputnik infection drastically alters the transcriptome of APMV rather than amoeba, likely by disturbing the transition from early to late stages of APMV infection.IMPORTANCEVirophages are small double-stranded DNA (dsDNA) viruses parasitizing other dsDNA viruses, such as giant viruses. Virophages inhibit the replication of giant viruses, and some protists use virophages as a defense system against giant viruses. However, molecular interactions among host cellular organisms, giant viruses, and virophages are largely unknown. Here, we performed a time-course transcriptome analysis of <i>Acanthamoeba castellanii</i> cells infected with Acanthamoeba polyphaga mimivirus (APMV) and those infected with both APMV and Sputnik 3 virophage. We demonstrated that the virophage has little effect on the amoeba transcriptome and primarily hijacks the transcriptional machinery of the giant virus. Furthermore, virophage infection alters giant virus gene expression, depending on their expression timing. The expression of early genes was prolonged, while that of late genes was delayed, suggesting that virophage infection disrupts the transition from the early to late stages of giant virus infection. This study provides molecular insights into the interactions within this unique tripartite system.