An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

Abstract

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. IMPORTANCEThe immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes.

The Role of Nuclear Antiviral Factors against Invading DNA Viruses: The Immediate Fate of Incoming Viral Genomes

“…BrdU (5-bromo-2’-deoxyuridine) is a classical analog widely used for DNA labeling, but requires stringent denaturing for detection using antibodies, similar to FISH. However, when combined with nuclease treatments, incorporated BrdU can be detected with antibodies without denaturing, and this method has been used to visualize viral genomes such as Ad DNAs [26]. …”

“…Based on this property, we recently developed a live-cell imaging technique, showing that EGFP-tagged TAF-I forms infection-specific nuclear puncta reflecting Ad genome localization in living cells [72]. Using these imaging tools, we have reported that incoming Ad genomes neither target nor are targeted by PML-NBs immediately after infection [26]. Furthermore, absence of co-localization between incoming Ad genomes and PML-NBs is neither cell type-specific nor reversed by IFN treatments, as has been shown by our and recent independent studies [26,27,73].…”

“…Using these imaging tools, we have reported that incoming Ad genomes neither target nor are targeted by PML-NBs immediately after infection [26]. Furthermore, absence of co-localization between incoming Ad genomes and PML-NBs is neither cell type-specific nor reversed by IFN treatments, as has been shown by our and recent independent studies [26,27,73]. These findings would be the first experimental evidence that incoming genomes of nuclear replicating DNA viruses are not necessarily trapped by PML-NBs upon nuclear entry, though it remains possible that these observations are related to specific experimental conditions (see Section 7).…”

“…E1A, an Ad immediate early gene product, was also shown to localize at PML-NBs [74]. However, incoming genomes of non-replicative, E1-deleted Ad vectors also do not localize at PML-NBs [26], making it unlikely that the expression of immediate early genes such as E1A prevents genome targeting by the bodies. In contrast, protein VI and IX, minor capsid proteins also present in incoming virions, have been reported to interact with PML-NBs and/or its components [77,78], which may affect the interplay between incoming Ad genomes and the bodies (discussed in Section 7).…”

“…By carefully reading the literature, however, we reach the conclusion that for many DNA viruses, the interplay between nuclear antiviral factors and incoming viral genomes immediately following genome import remains to be shown. Furthermore, our recent studies using adenovirus (Ad) go against the concept of direct genome detection by these nuclear factors, showing that freshly imported Ad genomes neither localize at PML-NBs nor are they targeted by its components or IFI16 upon nuclear entry [26,27]. In this review, we aim to provide a comprehensive description of nuclear antiviral mechanisms targeting viral genomes upon nuclear import in the immediate early phase of DNA virus infection, by listing existing experimental evidence and discussing conclusions that have been drawn from limited examples including our own.…”