Mimicry of Cellular A Kinase-Anchoring Proteins Is a Conserved and Critical Function of E1A across Various Human Adenovirus Species

Abstract

The E1A proteins of the various human adenovirus (HAdV) species perform the critical task of converting an infected cell into a setting primed for virus replication. While E1A proteins differ in both sequence and mechanism, the evolutionary pressure on viruses with limited coding capacity ensures that these proteins often have significant overlap in critical functions. HAdV-5 E1A is known to use mimicry to rewire cyclic AMP (cAMP) signaling by decoupling protein kinase A (PKA) from cellular A kinase-anchoring proteins (AKAPs) and utilizing PKA to its own advantage. We show here that E1As from other species of HAdV also possess this viral AKAP (vAKAP) function and examine how they manipulate PKA. E1A from most species of HAdV examined contain a small AKAP-like motif in their N terminus which targets the docking-dimerization domain of PKA as the binding interface for a conserved protein-protein interaction. This motif is also responsible for an E1A-mediated relocalization of PKA regulatory subunits from the cytoplasm into the nucleus, with species-specific E1A proteins having preference for one particular isoform of PKA subunit over another. Importantly, we showed that these newly characterized vAKAPs can integrate into cAMP-responsive transcription as well as contribute to viral genome replication and infectious progeny production for several distinct HAdV species.IMPORTANCE These data enhance the mechanistic knowledge on how HAdV E1A manipulates cellular PKA to benefit infection. The work establishes that mimicry of AKAPs and subversion of PKA-mediated cAMP signaling are conserved features for numerous human adenoviruses. This study also highlights the molecular determinants conferring selective protein-protein interactions between distinct PKA regulatory subunits and the different E1A proteins of these viruses. Additionally, it further emphasizes the utility of using viral proteins like E1A as tools for studying the molecular biology of cellular regulatory pathways.

Keywords: A-kinase anchoring protein; E1A; adenovirus; cyclic AMP; evolution; protein kinase A; protein-protein interaction; replication; viral AKAP; viral mimicry.

FIG 1

Protein kinase A subunits are conserved binding targets of HAdV E1A's N terminus. HT1080 cells were cotransfected with PKA subunits and various WT or mutant E1A constructs from 7 HAdV types (shown in the inset). Whole-cell lysates were harvested for coimmunoprecipitation. WT E1A from HAdV-3, -5, -9, -12, and -40 associates with RIα, RIIα, and Cα, whereas E1A from HAdV-4 and -52 does not. Deletion of the entire E1A N terminus ablated the interactions, whereas the N terminus alone was sufficient for binding.

FIG 2

E1A proteins that bind PKA use an AKAP-like sequence motif. (A to D) HT1080 cells were cotransfected with PKA subunits and E1A constructs from either HAdV-3, -9, -12, or -40. Mutation schemes are shown in the inset. Cell lysates were harvested, and coimmunoprecipitation analysis of each E1A protein revealed that amino acids 11 to 28 for HAdV-3 (A), aa 12 to 29 for HAdV-9 (B), aa 12 to 27, aa 37 to 49 for HAdV-12 (C), and aa 12 to 26 for HAdV-40 (D) were necessary and sufficient for binding PKA subunits.

FIG 3

HAdV species whose E1A proteins bind PKA do so via PKA's docking-dimerization domain. HT1080 cells were cotransfected with WT E1A from 7 species of HAdV along with either both WT PKA regulatory subunits (R) or both subunits lacking their D/D domains (ΔR). Whole-cell lysates were harvested for coimmunoprecipitation. Deletion of the D/D domains in either HA-tagged RIα or MYC-tagged RIIα abrogated the interactions with E1A.

FIG 4

Distinct E1A proteins relocalize PKA regulatory subunits in a type-specific manner. HeLa cells were transfected with EGFP-tagged WT or mutant E1A constructs from 7 species of HAdV. Cells were fixed and permeabilized, and proteins were detected via autofluorescence (E1A) or by immunofluorescence (PKA). Scale bars represent 100 μm, and the targeted PKA subunit is indicated above each group of panels. (A) EGFP alone or E1A from HAdV-4, -52, and -40 showed no relocalization of either PKA regulatory subunit. E1A from HAdV-3 and -12 showed no relocalization of RIα, whereas HAdV-5 and -9 E1A showed no relocalization of RIIα. (B) HAdV-5 and -9 E1A caused a shift of RIα from the cytoplasm to the nucleus that was ablated when these E1A proteins had their AKAP-like motifs deleted. (C) Conversely, HAdV-3 and -12 E1A caused a shift of RIIα from the cytoplasm to the nucleus that was ablated when their PKA-binding regions were removed. Quantification of nuclear signal relative to total cellular signal is also shown next to representative images for the indicated E1A species (values are displayed as means ± standard deviations; *, P < 0.001; n = 50).

FIG 5

C terminus of E1A, which contains the nuclear localization signal, is required for efficient retasking of PKA. A549 cells were infected with either HAdV-5 dl520 or dl1131/520 (the E1A proteins expressed by these viruses are shown in the adjacent panel). After 24 h, cells were fixed, permeabilized, and stained for confocal immunofluorescence. E1A lacking the C-terminal bipartite NLS was deficient for both its nuclear localization and recompartmentalization of RIα. Scale bars represent 100 μm.

FIG 6

Localization of PKA regulatory subunits in cells infected with various HAdV species. A549 cells were infected with WT HAdV-3, -4, -5 (dl309), -9, or -12 at an MOI of 5 (for HAdV-5) or 25 (for all other viruses). After 24 h cells were fixed, permeabilized, and stained for confocal immunofluorescence. RIα appears to be more nuclear in HAdV-5- or -9-infected cells than in uninfected cells. In contrast, RIIα appears more nuclear in HAdV-3- or -12-infected cells. Scale bars represent 100 μm.

FIG 7

Different E1A species display an affinity preference for either RIα or RIIα. (A) HT1080 cells were cotransfected with EGFP-tagged HAdV-5 E1A and PKA regulatory subunits. Untagged HAdV-5 E1A was titrated as a competitor against EGFP-E1A for binding PKA. Whole-cell lysates were harvested for co-IP using an antibody against EGFP. Increasing amounts of untagged E1A squelched EGFP-E1A's interactions with RIα and RIIα. (B) Conditions optimized from panel A were used in a larger competition panel with EGFP-tagged E1A from HAdV-3, -9, -12, and -40. HT1080 cells were similarly cotransfected with the indicated E1A species, PKA subunits, and increasing amounts of untagged HAdV-5 E1A. Co-IP analysis revealed various resistances and susceptibilities to competition from HAdV-5 E1A for binding either RIα or RIIα.

FIG 8

Depriving HAdV-5 E1A of type I PKA causes E1A to retask type II PKA instead. (A) A549 cells were treated with the indicated control or RIα-specific siRNAs and subsequently infected with WT (dl309) HAdV-5. Only in the absence of RIα protein does E1A relocalize some RIIα to the nucleus. Scale bars represent 100 μm. (B) Equilibrium models of how E1A may compete against cellular AKAPs (cAKAP) and determine which PKA isoform it relocalizes to the nucleus.

FIG 9

Viral AKAP mimicry is a modular function of E1A. (A) HT1080 cells were cotransfected with PKA subunits and either WT or chimeric E1A constructs, and cell lysates were harvested for co-IP. (Inset) Construction of the HAdV-4 and -5 E1A chimeras (the arrow indicates where N-terminal swap occurred). The HAdV-4 chimera gained the ability to bind PKA subunits, whereas this was lost for the HAdV-5 chimera. (B) HeLa cells were transfected with the indicated EGFP-tagged E1A constructs. Cells were fixed and permeabilized, and proteins were detected via autofluorescence (E1A) or by immunofluorescence (PKA). Cells transfected with WT HAdV-5 or the HAdV-4 chimera exhibited a shift of RIα from the cytoplasm into the nucleus. In contrast, WT HAdV-4 or the HAdV-5 chimera showed no noticeable relocalization of RIα. Scale bars represent 100 μm.

FIG 10

Specific residues in the AKAP-like motif of E1As induce preference for specific PKA subunits. (A) HT1080 cells were cotransfected with PKA subunits and the indicated E1A constructs, and cell lysates were harvested for co-IP. Both WT HAdV-3 and -5, along with mutants (HAdV-5 D21E, E26T, V27L and HAdV-3 E22D, T27E, L28V), retain similar levels of binding to PKA. The mutation scheme in the inset shows residue positions previously demonstrated as crucial in HAdV-5, along with the analogous positions in HAdV-3. (B) HeLa cells were transfected with the indicated EGFP-tagged E1A constructs. Cells were fixed and permeabilized, and proteins were detected via autofluorescence (E1A) or by immunofluorescence (PKA). Cells transfected with WT HAdV-3 or the HAdV-5 D21E, E26T, V27L mutant exhibited a shift of RIIα from the cytoplasm into the nucleus. In contrast, WT HAdV-5 or the HAdV-3 E22D, T27E, L28V mutant exhibited a relocalization of RIα. Scale bars represent 100 μm.

FIG 11

Interaction with PKA is required for full E1A-mediated transactivation of a PKA-driven promoter. HT1080 cells were transfected with a reporter consisting of the luciferase gene driven by the rat PEPCK promoter (sequence shown in the inset) along with various WT or mutant E1A constructs from 7 HAdV species. A 6-h treatment of 0.5 μM forskolin (fsk) and siRNA-mediated PKA knockdowns was performed to demonstrate this reporter's cAMP responsiveness and PKA-dependent regulation. Protein levels of each E1A construct are also shown. Luciferase activity was measured for each condition, and light units were set relative to an empty plasmid control. Each of the 7 WT E1As tested could transactivate this reporter, with those shown to bind PKA having the strongest effect. For the 5 species that interacted with PKA, deletion of their PKA-binding motifs conferred a statistically significant reduction on E1A's ability to induce this reporter (with the exception of HAdV-40). Values are displayed as means ± SEM. *, P < 0.05; n = 3.

FIG 12

HAdV-mediated stimulation of cAMP signaling allows WT E1A to retarget PKA activity. A549 cells were infected with either WT (dl309), ΔE1A (dl312), or E1AΔ4-25 (dl1101) HAdV-5 (MOI, 5) and examined at the indicated time points postinfection using confocal immunofluorescence (A and B), Western blotting (C), or kinase assay (D). (A) Cellular cAMP levels were examined before and after the onset of viral replication (as indicated by staining for HAdV-5 ssDNA-binding protein [DBP]). Production of cAMP is noticeably higher than that of uninfected cells both before and after E1A is expressed. Results with WT virus are shown as representative of all infected conditions. (B) Increased levels of phosphorylated CREB1 on Ser133 are present relative to uninfected cells and are shown for WT virus, localizing adjacent to and overlapping with virus replication centers. Scale bars represent 100 μm. (C) Western blot analysis of phosphorylated CREB1 detected increased levels under all infection conditions, which was highest in WT-infected cells after production of E1A protein. (D) Cell lysates were measured for PKA kinase activity and displayed similarly higher levels of activity under all infection conditions. Values are displayed as means ± SEM. *, P < 0.05; n = 3. (E) HT1080 cells were cotransfected with PKA regulatory and catalytic subunits along with HAdV-5 E1A. Co-IP analysis showed that E1A does not dissociate the PKA holoenzyme. (F) HT1080 cells were cotransfected with RIα and/or RIIα in the presence or absence of the HAdV-5 E1A N terminus and subjected to co-IP. No heterodimerization of PKA regulatory subunits was detectable even with a long-exposure (LE) Western blot.

FIG 13

Requirement of PKA for viral replication is conserved across numerous HAdV species. A549 cells were treated with control siRNA or siRNA specific for PKA subunits and infected with HAdV-5 (MOI, 5) or HAdV-3, -4, -9, or -12 (MOI, 25). Values for each virus were normalized to 1.0 in control-treated cells and are displayed as means ± SEM. n = 3. (A) DNA was isolated at 6 h postinfection as a gauge of viral input and at 48 h postinfection to measure viral genome replication. Relative viral DNA levels were quantified by qPCRs using a forward primer that recognizes a conserved sequence in E1A in combination with a species-specific reverse primer. (B) Cells were collected at 72 h postinfection, and production of infectious progeny virus was quantitatively assayed by plaque formation on HEK293 cells. For both genome replication and progeny production, knockdown of PKA regulatory subunits caused impairment for HAdV-3, -5, -9, and -12. The non-PKA binding HAdV-4 was unaffected. Knockdown of either the catalytic subunit or all 3 PKA components together impaired all viruses. *, P < 0.05; n = 3.