The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-β) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.

• Keywords
• cGAS sensor, immune sensing of DNA, mouse polyomavirus, p204 sensor, pattern recognition receptors,
• MeSH
• DNA, Viral genetics   immunology   MeSH
• Phosphoproteins antagonists & inhibitors   genetics   metabolism   MeSH
• Phosphorylation MeSH
• Tumor Virus Infections immunology   virology   MeSH
• Host-Pathogen Interactions MeSH
• Interferon-beta metabolism   MeSH
• Nuclear Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Membrane Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Mice MeSH
• Nucleotidyltransferases antagonists & inhibitors   genetics   metabolism   MeSH
• Polyomavirus Infections immunology   virology   MeSH
• Polyomavirus genetics   immunology   MeSH
• Immunity, Innate immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cGAS protein, mouse MeSH Browser
• DNA, Viral MeSH
• Phosphoproteins MeSH
• Ifi16 protein, mouse MeSH Browser
• Interferon-beta MeSH
• Nuclear Proteins MeSH
• Membrane Proteins MeSH
• Nucleotidyltransferases MeSH
• Sting1 protein, mouse MeSH Browser

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape.

• Keywords
• CAS9 gene editing, Cervical cancer, IFITM1, Interferon, MHC Class I molecule, SILAC mass spectrometry,
• MeSH
• Cell Line MeSH
• Antigens, Differentiation physiology   MeSH
• Humans MeSH
• Membrane Proteins physiology   MeSH
• Histocompatibility Antigens Class I metabolism   MeSH
• Uterine Cervical Neoplasms metabolism   MeSH
• RNA-Binding Proteins physiology   MeSH
• Protein Biosynthesis physiology   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Differentiation MeSH
• IFITM3 protein, human MeSH Browser
• leu-13 antigen MeSH Browser
• Membrane Proteins MeSH
• Histocompatibility Antigens Class I MeSH
• RNA-Binding Proteins MeSH

The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

• Keywords
• capsid proteins, importin β1, mouse polyomavirus, nuclear localization signal, trafficking into the nucleus,
• MeSH
• Biological Transport MeSH
• Cell Nucleus MeSH
• Cell Line MeSH
• DNA, Viral metabolism   MeSH
• Fluorescent Antibody Technique MeSH
• Nuclear Localization Signals genetics   MeSH
• Karyopherins metabolism   MeSH
• Mutation MeSH
• Mice MeSH
• Polyomavirus Infections metabolism   virology   MeSH
• Polyomavirus physiology   ultrastructure   MeSH
• Virus Assembly MeSH
• Amino Acid Substitution MeSH
• Protein Binding MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Viral MeSH
• Nuclear Localization Signals MeSH
• Karyopherins MeSH
• Capsid Proteins MeSH