Maarit Suomalainen

The role of the nuclear pore complex in adenovirus DNA entry

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA‐associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium‐depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O‐linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus‐binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.

The First Step of Adenovirus Type 2 Disassembly Occurs at the Cell Surface, Independently of Endocytosis and Escape to the Cytosol

ABSTRACT Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.

Human Immunodeficiency Virus Type 1 Assembly and Lipid Rafts: Pr55gag Associates with Membrane Domains That Are Largely Resistant to Brij98 but Sensitive to Triton X-100

ABSTRACT The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55 gag . We have analyzed whether Pr55 gag has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55 gag has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55 gag particles (VLPs) yield buoyant Pr55 gag complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55gag complexes cannot be taken as a proof for raft association of Pr55 gag , since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55 gag . However, Pr55 gag might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55 gag . Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55 gag complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55 gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55 gag VLPs.

Uncoating of non-enveloped viruses.

Non-enveloped viruses enclose their genome in capsids built of repetitive polypeptides interlinked with cementing proteins, divalent cations or disulphides. Interactions are broken in a stepwise manner during entry into cells leading to genome uncoating. Receptor or proteases induce conformational changes in case of rhinovirus, poliovirus or adenovirus, and thereby provide direct uncoating cues. Chemical cues from low endosomal pH activate rhinovirus or aphtovirus, and oxido-reductases mediate disulphide reshuffling of polyomavirus. Cellular motors provide a third class of cues as shown by adenoviruses. These examples highlight the diversity of cellular factors triggering virus uncoating, and offer new perspectives for the development of antivirals.

Vpu and Tsg101 Regulate Intracellular Targeting of the Human Immunodeficiency Virus Type 1 Core Protein Precursor Pr55gag

ABSTRACT Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55gag. Depending on the cell type, Pr55gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55gag.

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

ABSTRACT 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.

Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses

Viruses package their genome in a robust protein coat to protect it during transmission between cells and organisms. In a reaction termed uncoating, the virus is progressively weakened during entry into cells. At the end of the uncoating process the genome separates, becomes transcriptionally active, and initiates the production of progeny. Here, we triggered the disruption of single human adenovirus capsids with atomic force microscopy and followed genome exposure by single-molecule fluorescence microscopy. This method allowed the comparison of immature (noninfectious) and mature (infectious) adenovirus particles. We observed two condensation states of the fluorescently labeled genome, a feature of the virus that may be related to infectivity. Beyond tracking the unpacking of virus genomes, this approach may find application in testing the cargo release of bioinspired delivery vehicles.

Adenovirus Entry into Cells

Adenoviruses carry their DNA genome into post-mitotic nuclei of a variety of human cells, either within an organism (in vivo) or outside an organism in culture (ex vivo) (1). Recombinant adenoviruses are developed in many laboratories as gene delivery vehicles to treat hereditary and acquired human disorders of somatic cells (2; 3). Diseased lungs of cystic fibrosis patients have been pioneered for treatment with recombinant adenovirus vectors (4). Preliminary results are promising, but demonstrate that the disease has not yet been cured by the emerging gene transfer technology (5). One reason for limited success was that the transgenes were not expressed adequately in the diseased tissues, either due to low efficiency of virus delivery to the target cell or inefficient DNA import into the nucleus. In this chapter, we describe a quantitative method to determine transport of fluorescently labeled wild type adenovirus 2 to the nucleus of a model cell line, HeLa cells. This protocol should be directly applicable to recombinant adenoviruses in a variety of cell lines, including peripheral blood cells, fibroblasts, polarized epithelial cells and differentiated neurons.

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space I. BINDING TO DNA AND TO HEXON OF THE PRECURSOR TO PROTEIN VI, pVI, OF HUMAN ADENOVIRUS

Background: The C terminus of pVI activates the adenovirus proteinase. pVI escorts hexon into the nucleus. Results: pVI binds tightly to DNA independent of sequence, Kd = 46 nm. pVI binds tightly to hexon, Kd = 1.1 nm. Conclusion: DNA binding of pVI is the first step in the activation of adenovirus proteinase. Significance: This step links pVI, hexon, viral DNA, and the adenovirus proteinase in virion maturation. The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, Kd(app), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG00 of −4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its Kd(app) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl2 because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a Kd(app). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant Kd(app) of 1.1 nm.