The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome Release at the Nuclear Pore Complex.

Adenoviruses (AdVs) cause respiratory, ocular, and gastrointestinal tract infection and inflammation in immunocompetent people and life-threatening disease upon immunosuppression. AdV vectors are widely used in gene therapy and vaccination. Incoming particles attach to nuclear pore complexes (NPCs) of post-mitotic cells, then rupture and deliver viral DNA (vDNA) to the nucleus or misdeliver to the cytosol. Our genome-wide RNAi screen in AdV-infected cells identified the RING-type E3 ubiquitin ligase Mind bomb 1 (Mib1) as a proviral host factor for AdV infection. Mib1 is implicated in Notch-Delta signaling, ciliary biogenesis, and RNA innate immunity. Mib1 depletion arrested incoming AdVs at NPCs. Induced expression of full-length but not ligase-defective Mib1 in knockout cells triggered vDNA uncoating from NPC-tethered virions, nuclear import, misdelivery of vDNA, and vDNA expression. Mib1 is an essential host factor for AdV uncoating in human cells, and it provides a new concept for licensing virion DNA delivery through the NPC.

MxB is an interferon-induced restriction factor of human herpesviruses.

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid.

Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death.

Apoptosis and programmed necrosis (necroptosis) determine cell fate, and antagonize infection. Execution of these complementary death pathways involves the formation of receptor-interacting protein kinase 1 (RIPK1) containing complexes. RIPK1 binds to adaptor proteins, such as TRIF (Toll-IL-1 receptor-domain-containing-adaptor-inducing interferon-beta factor), FADD (Fas-associated-protein with death domain), NEMO (NF-κB regulatory subunit IKKγ), SQSTM1 (sequestosome 1/p62), or RIPK3 (receptor-interacting protein kinase 3), which are involved in RNA sensing, NF-κB signaling, autophagosome formation, apoptosis, and necroptosis. We report that a range of rhinoviruses impair apoptosis and necroptosis in epithelial cells late in infection. Unlike the double-strand (ds) RNA mimetic poly I:C (polyinosinic:polycytidylic acid), the exposure of dsRNA to toll-like receptor 3 (TLR3) in rhinovirus-infected cells did not lead to apoptosis execution. Accordingly, necroptosis and the production of ROS (reactive oxygen species) were not observed late in infection, when RIPK3 was absent. Instead, a virus-induced alternative necrotic cell death pathway proceeded, which led to membrane rupture, indicated by propidium iodide staining. The impairment of dsRNA-induced apoptosis late in infection was controlled by the viral 3C-protease (3Cpro), which disrupted RIPK1-TRIF/FADD /SQSTM1 immune-complexes. 3Cpro and 3C precursors were found to coimmuno-precipitate with RIPK1, cleaving the RIPK1 death-domain, and generating N-terminal RIPK1 fragments. The depletion of RIPK1 or chemical inhibition of its kinase at the N-terminus did not interfere with virus progeny formation or cell fate. The data show that rhinoviruses suppress apoptosis and necroptosis, and release progeny by an alternative cell death pathway, which is controlled by viral proteases modifying innate immune complexes.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection.

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein-protein VII. Two other Ad core proteins, V and X/µ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII-virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

Co-option of Membrane Wounding Enables Virus Penetration into Cells.

During cell entry, non-enveloped viruses undergo partial uncoating to expose membrane lytic proteins for gaining access to the cytoplasm. We report that adenovirus uses membrane piercing to induce and hijack cellular wound removal processes that facilitate further membrane disruption and infection. Incoming adenovirus stimulates calcium influx and lysosomal exocytosis, a membrane repair mechanism resulting in release of acid sphingomyelinase (ASMase) and degradation of sphingomyelin to ceramide lipids in the plasma membrane. Lysosomal exocytosis is triggered by small plasma membrane lesions induced by the viral membrane lytic protein-VI, which is exposed upon mechanical cues from virus receptors, followed by virus endocytosis into leaky endosomes. Chemical inhibition or RNA interference of ASMase slows virus endocytosis, inhibits virus escape to the cytosol, and reduces infection. Ceramide enhances binding of protein-VI to lipid membranes and protein-VI-induced membrane rupture. Thus, adenovirus uses a positive feedback loop between virus uncoating and lipid signaling for efficient membrane penetration.

A direct and versatile assay measuring membrane penetration of adenovirus in single cells.

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection.

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic dextran polymers. Thus, kinesin-1 uncoats viral DNA and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.

Drifting motions of the adenovirus receptor CAR and immobile integrins initiate virus uncoating and membrane lytic protein exposure.

Viral particle binding to plasma membrane receptors elicits virus motions, recruits signaling proteins, and triggers membrane bending and fission, finally resulting in endocytic virus uptake. Here we analyze how human adenovirus engages its receptor coxsackievirus adenovirus receptor (CAR) and coreceptor αv integrin to move on the plasma membrane. Virus binding to CAR through fiber knobs gave rise to diffusive motions and actomyosin-2-dependent drifts, while integrin-targeted viruses were spatially more confined. Diffusions, drifts, and confined motions were specifically observed with viral particles that were subsequently internalized. CAR-mediated drifts together with integrin binding supported fiber shedding from adenovirus particles, leading to exposure of the membrane-lytic internal virion protein VI and enhanced viral escape from endosomes. Our results show that adenovirus uncoating is initiated at the plasma membrane by CAR drifting motion and binding to immobile integrins.

Chemotactic antiviral cytokines promote infectious apical entry of human adenovirus into polarized epithelial cells.

Mucosal epithelia provide strong barriers against pathogens. For instance, the outward facing apical membrane of polarized epithelial cells lacks receptors for agents, such as hepatitis C virus, herpesvirus, reovirus, poliovirus or adenovirus. In addition, macrophages eliminate pathogens from the luminal space. Here we show that human adenovirus type 5 engages an antiviral immune response to enter polarized epithelial cells. Blood-derived macrophages co-cultured apically on polarized epithelial cells facilitate epithelial infection. Infection also occurs in the absence of macrophages, if virus-conditioned macrophage-medium containing the chemotactic cytokine CXCL8 (interleukin-8), or recombinant CXCL8 are present. In polarized cells, CXCL8 activates a Src-family tyrosine kinase via the apical CXCR1 and CXCR2 receptors. This activation process relocates the viral co-receptor ανß3 integrin to the apical surface, and enables apical binding and infection with adenovirus depending on the primary adenovirus receptor CAR. This paradigm may explain how other mucosal pathogens enter epithelial cells.

Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35.

Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here we show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, alphanu integrins, and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.