title: Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability.
abstract: A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication.
  
  
  
  The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.
authors.affiliation: Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
authors.forename: Matteo
authors.identifier: 0000-0001-7675-0762
authors.initials: M
authors.lastname: Biolatti
authors.affiliation: Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
authors.forename: Valentina
authors.identifier: 0000-0002-9336-7906
authors.initials: V
authors.lastname: Dell'Oste
authors.affiliation: Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Pautasso
authors.affiliation: Institute of Virology, University Hospital Ulm, Ulm, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: von Einem
authors.affiliation: Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Marschall
authors.affiliation: Institute for Virology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: Bodo
authors.initials: B
authors.lastname: Plachter
authors.affiliation: Department of Translational Medicine, Novara Medical School, Novara, Italy.
authors.forename: Marisa
authors.identifier: 0000-0002-5187-0140
authors.initials: M
authors.lastname: Gariglio
authors.affiliation: Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy Department of Translational Medicine, Novara Medical School, Novara, Italy.
authors.forename: Marco
authors.identifier: 0000-0002-3188-5783
authors.initials: M
authors.lastname: De Andrea
authors.affiliation: Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy santo.landolfo@unito.it.
authors.forename: Santo
authors.identifier: 0000-0002-3153-2865
authors.initials: S
authors.lastname: Landolfo
mesh_terms: D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000276:immunology* / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055550:Protein Stability; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D009687:Nuclear Proteins; D010750:Phosphoproteins; C109367:UL54 protein, Human herpesvirus 5; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C065418:cytomegalovirus matrix protein 65kDa; C076700:IFI16 protein, human; D004259:DNA-Directed DNA Polymerase