ISKNV-pORF71 hijacks outer mitochondrial membrane-localized VDAC2 to the nucleus, enhancing apoptosis by disrupting mitochondrial membrane permeability

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus within the family Iridoviridae, is one of the most devastating pathogens affecting global teleost populations. Our previous study confirmed that ISKNV-pORF71 (p71/VP71) is a viral virulence factor by constructing a recombinant virus with ISKNV orf071 deletion (ISKNV-{Delta}71). In the present study, we further found that compared with ISKNV-WT, the ISKNV-{Delta}71 mutant is more capable of maintaining mitochondrial membrane potential, alleviating mitochondrial membrane permeabilization, preserving mitochondrial membrane integrity, sustaining lower cytochrome c release, and thereby reducing the apoptosis rate of infected cells. Further investigations demonstrated a strong interaction between p71 and the outer mitochondrial membrane (OMM)-localized voltage-dependent anion channel 2 (VDAC2). At the subcellular level, p71 and mandarin fish VDAC2 (mfVDAC2) are originally localized to the nucleus and mitochondria, respectively. However, in cells co-transfected with p71 and mfVDAC2, robust nuclear translocation of mfVDAC2 was verified. Endogenous mfVDAC2 was also confirmed to undergo nuclear translocation upon ISKNV-WT infection. In the zebrafish infection model, zebrafish VDAC2 (zfVDAC2)--but not zfVDAC-1 or zfVDAC-3--was observed to translocate into the nucleus through interaction with p71. Using CRISPR/Cas9 technology, VDAC2-knockout zebrafish were successfully constructed. Infection experiments showed that ISKNV-{Delta}71 exhibits significantly reduced lethality to both wild-type zebrafish and zfVDAC2-knockout zebrafish. In conclusion, our findings reveal that p71 exerts its function during ISKNV infection by hijacking VDAC2 into the nucleus, which alters mitochondrial membrane permeability and enhances ISKNV-induced apoptosis. The phenotypic characteristics of VDAC2 knockout (vdac-/-) zebrafish were also thoroughly analyzed and discussed. Author summaryVDAC2, a well-characterized cellular protein localized to the outer mitochondrial membrane (OMM), plays critical roles in viral pathogenesis and antiviral immune escape. Previous studies have shown that VDAC2 interacts directly or indirectly with viral proteins or functions as a functional receptor to modulate viral pathogenesis; however, few studies have demonstrated that VDAC2 is hijacked from its native OMM to other organelles. Our study here provides robust evidence that cellular VDAC2 is rerouted from its original OMM to the nucleus through interaction with ISKNV p71, thereby altering viral pathogenesis. This work offers novel insights into the role of VDAC2 in pathogen virulence. As far as we know, this is the first report demonstrating that host VDAC2 is hijacked into the nucleus by a viral protein to subsequently modulate the viral life cycle. Collectively, these findings advance our understanding of the active regulation of host functional proteins by iridoviruses.