Transcriptome Analysis Reveals Gemykibivirus Infection Induces Mitochondrial DNA Release in HEK293T Cells

Gemykibivirus, an emerging single-stranded DNA (ssDNA) virus of the recently established genus in the family of Genomoviridae, had been discovered in human blood and cerebrospinal fluid and a variety of other body fluids. However, the molecular mechanisms of gemykibivirus entrance into the host cells and its pathogenicity remain poorly understood. To investigate the host response of gemykibivirus, we used an infectious clone of gemykibivirus previously established through molecular biology techniques to rescue virus in HEK293T cells and analyzed the changes in the host transcriptome during the infection period by RNA-Seq. Our findings indicate that gemykibivirus can both express viral proteins and accomplish replication, and high-throughput transcriptome analysis identified a total 1732 significantly different genes. Functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for differentially expressed genes (DEGs) showed gemykibivirus involving several important pathways, including MAPK signaling pathway, Chemical carcinogenesis-reactive oxygen species and Oxidative phosphorylation. Interestingly, mitochondrial DNA-encoded mRNAs exhibited varying levels of upregulation, suggesting that gemykibivirus may be involved in mitochondrial fission and the regulation of mitochondrial function. Subsequently, a series of experiments proved that gemykibivirus can lead an increase in mitochondrial DNA copy number, promote the release of mtDNA into the cytoplasm, enhance reactive oxygen species production and trigger other cellular antiviral responses. Overall, we lay a foundation for revealing the relationship between Gemykibivirus and human diseases through mitochondrial functional alterations.