Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1.

Adenovirus type 2 (Ad2) imports its DNA genome through the nuclear pore complex (NPC) of cells in interphase for viral production. Here we identify the NPC-filament protein CAN/Nup214 as a docking site for incoming Ad2 capsids. Binding to CAN is independent of cytosolic factors. Capsids disassemble at NPCs to free their DNA for import. This process requires binding of nuclear histone H1 to the stably docked capsids and involves H1-import factors, restricting this irreversible process to the proximity of the nucleus. Our results provide a molecular mechanism for disassembly of Ad2 and reveal an unexpected function of histone H1 in virus-mediated DNA import.

Shape reconstruction of subcellular structures from live cell fluorescence microscopy images.

Live imaging of subcellular structures is indispensible to advance our understanding of cellular processes. The blurred digital images acquired in light microscopy are, however, complex to analyze, and identification and reconstruction of subcellular structures from such images remains a major challenge. We present a novel, model-based image analysis algorithm to reconstruct outlines of subcellular structures using a sub-pixel representation. The algorithm explicitly accounts for the optical properties of the microscope. We validate the reconstruction performance on synthetic data and apply the new method to fluorescence microscopy images of endosomes identified by the GTPase EGFP-Rab5. The benefits of the new algorithm are outlined by comparison to standard techniques. We demonstrate that the new algorithm leads to better discrimination between different endosomal virus entry pathways and to more robust, accurate, and self-consistent quantification of endosome shape features. This allows establishing a set of features that quantify endosome morphology and robustly capture the dynamics of endosome fusion.

Nuclear targeting of SV40 and adenovirus.

Import o f viral DNA into the nucleus is essential for the successful replication o f DNA tumour viruses. To achieve this goal, viruses have adapted strategies to traverse the barriers between the plasma membrane and the nucleus o f a host cell. Two DNA tumour viruses, simian virus 40 and adenovirus, achieve the nuclear-entry step in slightly different ways. SV40 DNA enters the nucleus through the nuclear pore complexes (NPCs) in apparently intact virions. By contrast, adenovirus particles dissociate near the NPC before the viral DNA is imported into the nucleus. In both cases, karyophilic protein components o f the viruses appear to mediate nuclear entry o f the viral genomes. In this article, we discuss how an understanding o f the cell biology o f virus entry can help us understand the process o f nuclear transport.

Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein.

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion.

Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus.

Nuclear pore complexes provide channels for molecular transport across the nuclear envelope. Translocation of most proteins and RNAs through the pore complex is mediated by signal- and ATP-dependent mechanisms, while transport of small molecules is accomplished by passive diffusion. We report here that depletion of calcium from the lumen of the endoplasmic reticulum and nuclear envelope with ionophores or the calcium pump inhibitor thapsigargin rapidly and potently inhibits signal mediated transport of proteins into the nucleus. Lumenal calcium depletion also inhibits passive diffusion through the pore complex. Signal-mediated protein import and passive diffusion are rapidly restored when the drugs depleting lumenal calcium are removed and cells are incubated at 37 degrees C in calcium-containing medium. These results indicate that loss of calcium from the lumen of the endoplasmic reticulum and nuclear envelope reversibly affects properties of pore complex components located on the nuclear/cytoplasmic membrane surfaces, and they provide direct functional evidence for conformational flexibility of the pore complex. These methods will be useful for achieving reversible inhibition of nucleocytoplasmic trafficking for in vivo functional studies, and for studying the structure of the passive diffusion channel(s) of the pore complex.

Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus.

Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

Nuclear import in viral infections.

The separation of transcription in the nucleus and translation in the cytoplasm requires nucleo-cytoplasmic exchange of proteins and RNAs. Viruses have evolved strategies to capitalize on the nucleo-cytoplasmic trafficking machinery of the cell. Here, we first discuss the principal mechanisms of receptor-mediated nuclear import of proteinaceous cargo through the nuclear pore complex, the gate keeper of the cell nucleus. We then focus on viral strategies leading to nuclear import of genomes and subgenomic particles. Nucleo-cytoplasmic transport is directly important for those viruses that are replicating in the nucleus, such as DNA tumor viruses and RNA viruses, including parvoviruses, the DNA retroviruses hepadnaviruses, RNA-retrotransposons and retroviruses, adenoviruses, herpesviruses, papovaviruses, and particular negative-sense RNA viruses, such as the orthomyxovirus influenza virus. The viral strategies of nuclear import turn out to be surprisingly diverse. Their investigation continues to give insight into how nucleic acids pass in and out of the nucleus.

Mechanisms of virus uncoating.

In a virus particle, the genome is highly condensed and protected by proteins and membrane bilayers. Before it can be replicated in a new host cell, uncoating must take place. Recent studies on enveloped and nonenveloped animal viruses indicate that uncoating occurs through complex, multistep processes triggered by virus-host-cell interactions.

Galactosyltransferase-dependent sialylation of complex and endo-N-acetylglucosaminidase H-treated core N-glycans in vitro.

Purified beta-N-acetylglucosaminide beta(1-4)galactosyltransferase and partially purified beta-galactoside alpha(2-6)-sialyltransferase were used to elongate and terminate glycan chains of agalacto-ovalbumin and endo-beta-N-acetylglucosaminidase H-treated yeast invertase in vitro. In the presence of both transferases, 0.1 mol sialic acid was incorporated per mol agalacto-ovalbumin within 24 h. Evidence is presented to show that purification of the galactosylated intermediate increases the efficiency of sialylation. Incorporation of sialic acid into endo-beta-N-acetylglucosaminidase H-treated oligomannose glycoproteins may be useful for in vivo stabilization of these glycoproteins by preventing uptake in liver or reticuloendothelial cells.

Purification of endo-N-acetyl-beta-D-glucosaminidase H by substrate-affinity chromatography.

Endo-N-acetyl-beta-D-glucosaminidase H (Endo H) was purified to homogeneity (3000-fold) from a culture filtrate to Streptomyces plicatus. The key step was substrate-affinity chromatography, which afforded a 1000-fold purification and yielded a protease- and exoglycosidase-free preparation of Endo H. Proteins from the crude sample were applied to the substrate-affinity column, consisting of yeast-invertase glycopeptides bound to Sepharose-immobilized concanavalin A. After washing off the unbound proteins, Endo H was quantitatively eluted by methyl alpha-D-mannopyranoside. Various conditions were tested to achieve an optimal binding of Endo H to this substrate-affinity gel. After substrate-affinity chromatography, Endo H was separated from the coeluted gylcopeptide substrate and some protein impurities by gel filtration and hydrophobic chromatography.

Adenovirus-triggered innate signalling pathways.

Adenoviruses are important infectious agents and also emerging vectors in different biomedical applications. These viruses elicit a strong innate and adaptive immune response, which influences both the course of disease and the success of the applied vectors. Several Toll-like Receptor (TLR)-dependent and -independent mechanisms contribute to these responses. Understanding of the involved viral and cellular factors is crucial for the treatment of various adenovirus diseases and the optimal design of adenovirus vector applications. Here we summarize our current understanding of the complex nature of adenovirus-induced innate immune mechanisms.

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Signalling in viral entry.

Viral infections are serious battles between pathogens and hosts. They can result in cell death, elimination of the virus or latent infection keeping both cells and pathogens alive. The outcome of an infection is often determined by cell signalling. Viruses deliver genomes and proteins with signalling potential into target cells and thereby alter the metabolism of the host. Virus interactions with cell surface receptors can elicit two types of signals, conformational changes of viral particles, and intracellular signals triggering specific cellular reactions. Responses by cells include stimulation of innate and adaptive immunity, growth, proliferation, survival and apoptosis. In addition, virus-activated cell signalling boosts viral entry and gene delivery, as recently shon for adenoviruses and adeno-associated viruses. This review illustrates that multiple activation of host cells during viral entry profoundly impacts the elaborate relationship between hosts and viral pathogens.

Quantitative microscopy of fluorescent adenovirus entry.

Fluorescence imaging of cells is a powerful tool for exploring the dynamics of organelles, proteins, and viruses. Fluorescent adenoviruses are a model system for cargo transport from the cell surface to the nucleus. Here, we describe a procedure to quantitate adenovirus-associated fluorescence in different subcellular regions. CCD camera-captured fluorescence sections across entire cells were deblurred by a fast Fourier transformation, the background was subtracted images merged, and virus fluorescence quantitated. The validity of the deblurring routine was verified by confocal laser scanning microscopy, demonstrating that objects were neither generated nor deleted. Instead, the homogeneity of both the average intensity and the size of fluorescent particles was increased, facilitating automated quantification. We found that nuclear fluorescence of wt adenovirus, but not of a virus mutant ts1, which fails to escape from endosomes, was maximal at 90 min postinfection (p.i.). Surprisingly, nuclear fluorescence decreased at 120 min, but increased again at 240 min p.i., suggesting that wt virus targeting to the nucleus may be multiphasic and regulated. Interestingly, only the first nuclear transport period of wt but not ts1 virus coincided with a significant increase of the peripheral and decrease of the cytoplasmic regions, indicative of signal-dependent cell contraction.

A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail.

One of a small number of polypeptides of the nuclear pore complex that have been identified is a major glycoprotein called gp210. Since it is very resistant to chemical extractions from membranes, gp210 was suggested to be integrated into nuclear membranes. In this study we have determined the membrane topology of this protein by biochemical and immunological approaches. We found that limited proteolysis of isolated nuclear envelopes with papain released a 200 kd water-soluble fragment of gp210 containing concanavalin A-reactive carbohydrate. Immunogold electron microscopy with a monoclonal antibody showed that this domain is localized on the lumenal side of nuclear membranes at pore complexes. Anti-peptide antibodies against two sequences near the C-terminus of gp210 were used to map possible membrane spanning and cytoplasmically disposed regions of this protein. From analysis of the protease sensitivity of these epitopes in sealed membrane vesicles, we determined that gp210 contains a small cytoplasmic tail and only a single membrane-spanning region. Thus, gp210 is a transmembrane protein with most of its mass, including the carbohydrate, located in the perinuclear space. This topology suggests that gp210 is involved primarily in structural organization of the pore complex, for which it may provide a membrane attachment site.

Stepwise dismantling of adenovirus 2 during entry into cells.

Adenoviruses enter their host cells by receptor-mediated endocytosis and acid-activated penetration from endosomes into the cytosol and deliver their DNA genome into the nucleus. Our results show that incoming adenovirus type 2 particles undergo a stepwise disassembly program necessary to allow progress of the virus in the entry pathway and release of the genome into the nucleus. The fibers are released, the penton base structures dissociated, the proteins connecting the DNA to the inside surface of the capsid degraded or shed, and the capsid-stabilizing minor proteins eliminated. The uncoating process starts immediately upon endocytic uptake with the loss of fibers and ends with the uptake of dissociated hexon proteins and DNA into the nucleus.

The role of the adenovirus protease on virus entry into cells.

Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid-stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re-entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry-competent virus but also to disassemble the incoming virus.

Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus.

Nuclear targeting of adenovirus is mediated by the microtubule-dependent, minus-end-directed motor complex dynein/dynactin, in competition with plus- end-directed motility. We demonstrate that adenovirus transiently activates two distinct signaling pathways to enhance nuclear targeting. The first pathway activates integrins and cAMP-dependent protein kinase A (PKA). The second pathway activates the p38/MAP kinase and the downstream MAPKAP kinase 2 (MK2), dependent on the p38/MAPK kinase MKK6, but independent of integrins and PKA. Motility measurements in PKA-inhibited, p38-inhibited or MK2-lacking (MK2(-/-)) cells indicate that PKA and p38 stimulated both the frequency and velocity of minus-end-directed viral motility without affecting the perinuclear localization of transferrin-containing endosomal vesicles. p38 also suppressed lateral viral motilities and MK2 boosted the frequency of minus-end-directed virus transport. Nuclear targeting of adenovirus was rescued in MK2(-/-) cells by overexpression of hsp27, an MK2 target that enhances actin metabolism. Our results demonstrate that complementary activities of PKA, p38 and MK2 tip the transport balance of adenovirus towards the nucleus and thus enhance infection.

The alphavbeta5 integrin of hematopoietic and nonhematopoietic cells is a transduction receptor of RGD-4C fiber-modified adenoviruses.

Epithelial and endothelial cells expressing the primary Coxsackie virus B adenovirus (Ad) receptor (CAR) and integrin coreceptors are natural targets of human Ad infections. The fiber knob of species A, C, D, E and F Ad serotypes binds CAR by mimicking the CAR-homodimer interface, and the penton base containing arginine-glycine-aspartate (RGD) motifs binds with low affinity to alphav integrins inducing cell activation. Here, we generated seven different genetically modified Ad vectors with RGD sequences inserted into the HI loop of fiber knob. All mutants bound and infected CAR and alphav integrin-positive epithelial cells with equal efficiencies. However, the Ads containing two additional cysteines, both N and C terminals of the RGD sequence (RGD-4C), were uniquely capable of transducing CAR-less hematopoietic and nonhematopoietic human tumor cell lines and primary melanoma cells. Both binding and transduction of RGD-4C Ad were blocked by soluble RGD peptides. Flow cytometry of cell surface integrins and virus binding to CAR-less cells in the presence of function-blocking anti-integrin antibodies indicated that the alphavbeta5 integrin, but not alphavbeta3, alphaIIbbeta3 or beta1,alpha5 or alpha6-containing integrins served as a functional transduction receptor of the RGD-4C Ads. However, in cells with low levels of alphavbeta5 integrin, the function-blocking anti-alphavbeta5 antibodies were not effective, unlike soluble RGD peptides. Collectively, our data demonstrate that the alphavbeta5 integrin is a functional transduction receptor of RGD-4C Ads in the absence of CAR, and that additional RGD receptors are targets of these viruses. The RGD-4C vectors further extend the tropism of Ads towards potential human therapies.

Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals.

The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy metal-induced transcription of metallothionein I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF-1 is mediated through binding to metal-responsive elements of consensus TGCRCNC in the target gene promoters. In an attempt to further clarify the mechanisms by which certain external signals activate MTF-1 and in turn modulate gene transcription, we show here that human MTF-1 has a dual nuclear and cytoplasmic localization in response to diverse stress stimuli. MTF-1 contains a consensus nuclear localization signal located just N-terminal to the first zinc finger that contributes to but is not essential for nuclear import. MTF-1 also harbors a leucine-rich, nuclear export signal. Under resting conditions, the nuclear export signal is required for cytoplasmic localization of MTF-1 as indicated by mutational analysis and transfer to the heterologous green fluorescent protein. Export from the nucleus was inhibited by leptomycin B, suggesting the involvement of the nuclear export protein CRM1. Our results further show that in addition to the heavy metals zinc and cadmium, heat shock, hydrogen peroxide, low extracellular pH (pH 6.0), inhibition of protein synthesis by cycloheximide, and serum induce nuclear accumulation of MTF-1. However, heavy metals alone (and not the other stress conditions) induce a significant transcriptional response via metal-responsive element promoter sequences, implying that nuclear import of MTF-1 is necessary but not sufficient for transcriptional activation. Possible roles for nuclear import under non-metal stress conditions are discussed.