Adenoviral protein E4orf4 interacts with the polarity protein Par3 to induce nuclear rupture and tumor cell death

Abstract

The tumor cell–selective killing activity of the adenovirus type 2 early region 4 ORF4 (E4orf4) protein is poorly defined at the molecular level. Here, we show that the tumoricidal effect of E4orf4 is typified by changes in nuclear dynamics that depend on its interaction with the polarity protein Par3 and actomyosin contractility. Mechanistically, E4orf4 induced a high incidence of nuclear bleb formation and repetitive nuclear ruptures, which promoted nuclear efflux of E4orf4 and loss of nuclear integrity. This process was regulated by nucleocytoskeletal connections, Par3 clustering proximal to nuclear lamina folds, and retrograde movement of actin bundles that correlated with nuclear ruptures. Significantly, Par3 also regulated the incidence of spontaneous nuclear ruptures facilitated by the downmodulation of lamins. This work uncovered a novel role for Par3 in controlling the actin-dependent forces acting on the nuclear envelope to remodel nuclear shape, which might be a defining feature of tumor cells that is harnessed by E4orf4.

The FDA-Approved Drug Nelfinavir Inhibits Lytic Cell-Free but Not Cell-Associated Nonlytic Transmission of Human Adenovirus

“…This is in agreement with the observation that HAdV types of the A, B, and D species form comet-shaped plaques and that ADP-deleted HAdV-C2 lyses the host cell and forms comet-shaped plaques albeit delayed and with lower efficacy than ADP-containing rec700 or HAdV-C2-dE3B-GFP. Conspicuously, other AdV proteins besides ADP were reported to interfere with cell lysis, such as the early region 4 open reading frame 4 (ORF4) protein, which induces nuclear envelope blebbing and promotes the loss of nuclear integrity ( 145 , 146 ). This, together with diverse cellular mechanisms underlying force generation and membrane rupture, could compensate for the lack of ADP in some forms of lytic virus egress ( 51 , 55 , 146 ).…”

“…Conspicuously, other AdV proteins besides ADP were reported to interfere with cell lysis, such as the early region 4 open reading frame 4 (ORF4) protein, which induces nuclear envelope blebbing and promotes the loss of nuclear integrity ( 145 , 146 ). This, together with diverse cellular mechanisms underlying force generation and membrane rupture, could compensate for the lack of ADP in some forms of lytic virus egress ( 51 , 55 , 146 ). We consider it unlikely that genetic variability of the inoculum accounts for the presence of lytic and nonlytic pathways since the inoculum was derived from an infectious DNA clone of HAdV-C2-dE3B-GFP and lacked any mutations affecting amino acid coding across many passages ( 72 ).…”

Chaperone-Assisted Mitotic Actin Remodeling by BAG3 and HSPB8 Involves the Deacetylase HDAC6 and Its Substrate Cortactin

“…DNA was stained with cell permeable Hoechst (150 ng/mL). Specimens were processed for immunofluorescence as described [ 55 ] and were post-fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. The epifluorescence images were acquired on a AxioObserver Z1 system using a 60x 1.25NA objective or a 40x Plan-Neofluoar 0.6 NA objective and a charged coupled device (CCD) camera Axiocam MRm controlled by the Zen software (ZEISS Group, Baden-Wurttenberg, Germany).…”

EPH Receptor Tyrosine Kinases Regulate Epithelial Morphogenesis and Phosphorylate the PAR-3 Scaffold Protein to Modulate Downstream Signaling Networks

“…For stable expression in mammalian cells, human sequences of EPHA4, EPHB2, EPHB3, EPHB4 were cloned into pcDNA5-FRT-TO-BirA*-FLAG or pcDNA5-FRT-TO vectors (Thermo Fisher Scientific) and human EFNB1 and EFNB2 were subcloned into pcDNA5-FRT-TO with a C-terminal 2xHA tag. Previously described murine EPHA4 WT and KD in pcDNA3.1(-) (Dionne et al 2018), rat PARD3 cDNA in pEGFP-C1 (Hidalgo-Carcedo et al 2011), and PARD3 domain deletion mutants (Dziengelewski et al 2020) were used for transient expression. Human sequences of EPHA1, EPHA2, EphA4 and EphB4 were cloned into pLJM1-EGFP vector (Addgene plasmid # 19319, from David Sabatini) for transient infections.…”

“…Individual GFP positive clones were picked, expanded and analyzed for EPHB2 expression. HEK293T cells stably expressing shPar3 described in (Dziengelewski et al 2020) were grown in the presence of 1.5 mg.ml −1 puromycin. Transient transfections of HEK293T and HEK293T-shPar3 cells were performed using polyethylenimine (PEI).…”