Early stage IFI16 cytoplasmic translocation and late stage entrapment into egressing virions during HCMV infection

Intrinsic immune mechanisms constitute frontline antiviral defense mediated by constitutively expressed proteins termed “restriction factors”. We recently demonstrated that the DNA sensor IFI16 restricts HCMV replication by down-regulating viral early and late but not immediate-early mRNAs and their protein expression. Here, we show that at an early time point during the in vitro infection of low-passage human embryonic lung fibroblasts (HELF) IFI16 binds to HCMV DNA. However, at a later phase following infection, IFI16 is mislocalized to the cytoplasmic assembly complex (AC), where it colocalizes with viral structural proteins. Upon pUL97 binding, indeed, IFI16 undergoes phosphorylation and relocalizes into the cytoplasm of HCMV-infected cells. ESCRT (Endosomal Sorting Complex Required for Transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blot analysis of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16 into mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established.