As obligate intracellular parasites, viruses are dependent on their infected hosts for survival. Consequently, viruses are under enormous selective pressure to utilize available cellular components and processes to their own advantage. As most, if not all, cellular activities are regulated at some level via protein interactions, host protein interaction networks are particularly vulnerable to viral exploitation. Indeed, viral proteins frequently target highly connected “hub” proteins to “hack” the cellular network, defining the molecular basis for viral control over the host. This widespread and successful strategy of network intrusion and exploitation has evolved convergently among numerous genetically distinct viruses as a result of the endless evolutionary arms race between pathogens and hosts. Here we examine the means by which a particularly well-connected viral hub protein, human adenovirus E1A, compromises and exploits the vulnerabilities of eukaryotic protein interaction networks. Importantly, these interactions identify critical regulatory hubs in the human proteome and help define the molecular basis of their function.

DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation.

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease, however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1 mediated nuclear import. IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms, however individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro. This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of Imp-α to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs.

No abstract