Innate nuclear sensor IFI16 translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage.
Intrinsic immune mechanisms mediated by constitutively expressed proteins termed "restriction factors" provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to 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 blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established.


OBJECTIVE
Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
Valentina
V
Dell'Oste
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
Deborah
D
Gatti
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
Francesca
F
Gugliesi
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy Department of Translational Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy.
Marco
M
De Andrea
Department of Translational Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy.
Mandar
M
Bawadekar
Department of Translational Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy.
Irene
I
Lo Cigno
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
Matteo
M
Biolatti
Institute of Plant Virology, National Research Council, Turin, Italy.
Marta
M
Vallino
Institute for Clinical and Molecular Virology, Division of Biotechnology, University of Erlangen-Nuremberg, Erlangen, Germany.
Manfred
M
Marschall
Department of Translational Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy.
Marisa
M
Gariglio
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy santo.landolfo@unito.it.
Santo
S
Landolfo
D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D003593:Cytoplasm / Q000378:metabolism* / Q000821:virology; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000502:physiology*; D057074:Virus Release; D014779:Virus Replication
D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; D010750:Phosphoproteins; D015678:Viral Structural Proteins; C076700:IFI16 protein, human