Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. We found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin "tail" formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.

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.

Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential.DNA viruses and retroviruses maintain and replicate their genomes in host cell nuclei by using histone-based nucleosomes, similar to chromatin, or they encode their own DNA binding and DNA-organizing proteins (34,45,47). They assemble and maintain their genomes in different chromatin states by packaging the nucleic acids into proteinaceous capsids and sometimes lipid envelopes and thereby traffic their genome within and transmit it between cells (8, 41). The simian virus 40 (SV40) polyomavirus, for example, packages its virion DNA with cellular core histones and uses histones to replicate in infected nuclei (19). Herpesviruses, on the other hand, condense their double-strand DNA in particles with the help of polyamines and use histones during latent residence within infected nuclei or use irregularly spaced nucleosomes during productive phases of infection (45).Adenoviruses (Ads) replicate and assemble particles in the nucleus. They encode their own histone-like proteins to condense a linear double-strand DNA genome of about 36 kbp into a proteinaceous DNA core. Although it is unknown how the viral DNA is precisely organized in the virion, isolated cores of species C human adenovirus serotypes 2 and 5 (Ad2/5) contain six viral proteins, the basic proteins V (pV), pVII, and pX; the terminal protein covalently attached to the 5Ј ends of the DNA; and small numbe...

Human adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C adenoviruses Ad2 and Ad5 (Ad2/5) cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L) in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC) gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein), which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for genetic analyses of distinct host requirements for Ad endocytosis and escape from endosomes.

DNA-tumor viruses comprise enveloped and non-enveloped agents that cause malignancies in a large variety of cell types and tissues by interfering with cell cycle control and immortalization. Those DNA-tumor viruses that replicate in the nucleus use cellular mechanisms to transport their genome and newly synthesized viral proteins into the nucleus. This requires cytoplasmic transport and nuclear import of their genome. Agents that employ this strategy include adenoviruses, hepadnaviruses, herpesviruses, and likely also papillomaviruses, and polyomaviruses, but not poxviruses which replicate in the cytoplasm. Here, we discuss how DNA-tumor viruses enter cells, take advantage of cytoplasmic transport, and import their DNA genome through the nuclear pore complex into the nucleus. Remarkably, nuclear import of incoming genomes does not necessarily follow the same pathways used by the structural proteins of the viruses during the replication and assembly phases of the viral life cycle. Understanding the mechanisms of DNA nuclear import can identify new pathways of cell regulation and anti-viral therapies.

Imaging across scales reveals disease mechanisms in organisms, tissues, and cells. Yet, particular infection phenotypes, such as virus-induced cell lysis, have remained difficult to study. Here, we developed imaging modalities and deep learning procedures to identify herpesvirus and adenovirus (AdV) infected cells without virusspecific stainings. Fluorescence microscopy of vital DNA-dyes and live-cell imaging revealed learnable virus-specific nuclear patterns transferable to related viruses of the same family. Deep learning predicted two major AdV infection outcomes, nonlytic (nonspreading) and lytic (spreading) infections, up to about 20 hr prior to cell lysis. Using these predictive algorithms, lytic and non-lytic nuclei had the same levels of green fluorescent protein (GFP)-tagged virion proteins but lytic nuclei enriched the virion proteins faster, and collapsed more extensively upon laser-rupture than non-lytic nuclei, revealing impaired mechanical properties of lytic nuclei. Our algorithms may be used to infer infection phenotypes of emerging viruses, enhance single cell biology, and facilitate differential diagnosis of non-lytic and lytic infections.

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artificial intelligence | machine learning | deep learning | predictive infection | computational image analyses | single cell analyses | nucleus | live cell DNA stain | nuclear rupture | nuclear pressure | nuclear envelope | live cell imaging | fluorescence microscopy | laser ablation | adenovirus | herpes simplex virus | virus spreading | virus egress | oncolytic virus | cell lysis | lytic infectionCorrespondence: urs.greber@imls.uzh.ch