Getting it together in plant virus movement: cooperative interactions between bipartite geminivirus movement proteins

To move cell-to-cell and systemically infect the host, plant viruses must cross the barrier posed by the plant cell wall. Plant viruses accomplish this through strategies that alter the architecture of the infected cell, eliminating this barrier through the action of viral-encoded 'movement proteins'. Detailed studies of a number of cytoplasmically replicating viruses suggest that movement proteins interact with components of the cytoskeleton and transport systems of the plant cell to allow passage of progeny into adjacent cells. Recent work on the two movement proteins encoded by the phloem-restricted geminivirus squash leaf curl virus has defined unique aspects of nuclear transport and protein protein interaction in the movement of this nuclear-replicating virus, and suggests that post-translational phosphorylation may be important in the regulation of movement protein function.     

The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles.

Specific premessenger ribonucleoprotein (pre-mRNP) particles, the Balbiani ring (BR) granules in the salivary glands of the dipteran Chironomus tentans, can be visualized in the electron microscope when they assemble on the genes, move through nucleoplasm, and bind to and translocate through the nuclear pores. As shown by BrUTP labeling and immunoelectron microscopy, newly synthesized BR RNP particles, released from the BR genes, appear early in all nucleoplasmic regions of the cell nucleus and they saturate the nucleoplasmic pool of BR particles after 2 h of labelling. It is concluded that within the nucleus the BR particles move randomly. Furthermore, estimates of minimum diffusion coefficients for the BR particles are compatible with the view that the particles diffuse freely in the interchromosomal space, although it is not excluded that the random movement could be slightly retarded. Once the particles get bound to the nuclear pore complexes, they seem committed to translocation through the nuclear pores.     

Structure of the nuclear pore complex in mammalian cells. Two annular components

The ultrastructure of the nuclear pore complex has been investigated in isolated nuclei of an in vitro cultured bovine liver cell line. In shadow-cast replicas of the surface of nuclei isolated in Tris buffer containing low K⁺ and Mg²⁺ concentrations (RSB) the rims of the pores appeared as annular projections with an outer diameter of 100 to 120 nm. When the nuclei were isolated in Tris buffer containing 0.1% Triton the projections were essentially lost, together with the outer membrane of the nuclear envelope. In electron micrographs of whole-mount preparations the Triton-Tris nuclei—but not the RSB nuclei—were surrounded by numerous circular structures, which obviously had been detached from the nuclear surface during the preparation. They consisted of eight granules of about 20 nm diameter which were connected in a circular fashion by fibrous material. The circular structures had an inside diameter close to 65 nm. In broken nuclei many of these circular structures contained a second, smaller circular component and a central granule. From these observations it is concluded that the annulus of the nuclear pore consists of two components and that the outer component is located in the perinuclear space in intimate association with the membrane limiting the pore. A modified model of the nuclear pore complex which accounts for this location is proposed.     

ATP-Induced Shape Change of Nuclear Pores Visualized with the Atomic Force Microscope

Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mm) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-γ-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore ``contraction.' Received: 10 February 1997/Revised: 10 February 1998     

Ultrastructure of fat body cells of the velvetbean caterpillar infected with a nuclear polyhedrosis virus

During a study of the ultrastructure of a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis, various types of nuclear and cytoplasmic inclusions were found in fat body tissue heavily infected with the virus. Virogenic stroma was present in the nuclei of most infected cells. Bundles of fibrous material were observed in the nuclei and cytoplasm of cells containing polyhedral bodies. Other nuclear inclusions included concentric multilayered material, vacuoles, and membrane structures.     

Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures.

Herpes simplex virus DNA replication proteins localize in characteristic patterns corresponding to viral DNA replication structures in the infected cell nucleus. The intranuclear spatial organization of the HSV DNA replication structures and the factors regulating their nuclear location remain to be defined. We have used the HSV ICP8 DNA-binding protein and bromodeoxyuridine labeling as markers for sites of herpesviral DNA synthesis to examine the spatial organization of these structures within the cell nucleus. Confocal microscopy and three-dimensional computer graphics reconstruction of optical series through infected cells indicated that viral DNA replication structures extend through the interior of the cell nucleus and appear to be spatially separate from the nuclear lamina. Examination of viral DNA replication structures in infected, binucleate cells showed similar or virtually identical patterns of DNA replication structures oriented along a twofold axis of symmetry between many of the sister nuclei. These results demonstrate that HSV DNA replication structures are organized in the interior of the nucleus and that their location is defined by preexisting host cell nuclear architecture, probably the internal nuclear matrix.     

Increased Larval Growth and Preference for Virus-Infected Leaves by the Mexican Bean Beetle, Epilachna varivestis Mulsant, a Plant Virus Vector

Phaseolus vulgaris L. cv. 'Black Valentine' is a systemic host for the plant viruses Southern bean mosaic virus (SBMV) and bean pod mottle virus (BPMV). The Mexican bean beetle, Epilachna varivestis Mulsant, is a vector of SBMV and BPMV. Our objective was to determine if the interaction of SBMV and BPMV with 'Black Valentine' bean plants would affect beetle behavior and growth. In adult feeding preference test assays, beetles preferred and ingested more of the virus-infected bean leaf tissue than the noninfected leaf tissue. Beetle larvae that fed on SBMV- or BPMV-infected plants weighed more than those that fed on healthy plants. Our experiments suggest that there might be a mutually beneficial relationship between the beetle and the viruses that it vectors. The virus benefits from being transmitted and the beetle benefits from better larval growth when feeding on virus-infected leaf tissue. This study further demonstrates the complexity of relationships between multiple organisms.     

The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells

The polypeptide species of non-membranous nuclear ghosts from purified cell nuclei are conserved among a variety of human, hamster and mouse cell types studied, including HeLa, BHK, 3T6 and Hep-2 cell lines. The polypeptide species present in nuclear ghosts from HeLa cells synchronized in various stages of the cell cycle are largely the same with minor variations. The isolated nuclear ghosts are similar, in terms of polypeptide composition, to other residual nuclear structures isolated by independent techniques. The nuclear ghosts appear as flattened sac-like structures when viewed by scanning electron microscopy. Transmission electron microscopy of the nuclear ghosts reveals ring-like structures which may represent the nuclear pores. Also observed are novel rod-shaped structures approximately 260 nm in length and 50 nm in diameter. The latter images either arise by a rearrangement during isolation of the nuclear ghost macromolecules or are a heretofore undescribed structure of intact nuclei.     

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.     

Simian virus 40 associates with nuclear superstructures at early times of infection.

The association of infecting simian virus 40 with insoluble nuclear structures was assayed by disrupting infected nuclei and assaying insoluble fractions for virus. Three methods were used which lyse nuclei but maintain the insolubility of residual nuclear structures: sonication, high-salt-Triton-EDTA extraction, and low-salt-lithium diiodosalicylate extraction. After each type of nuclear extraction, infecting simian virus 40 remained associated with the residual nuclear structures. This association depended strictly on natural viral infections and on the use of buffers containing moderate amounts of salt and Mg2+ for the isolation of infected nuclei. These viral interactions exhibited behavior similar to host cell DNA interactions studied by analogous assays. Both viral DNA and coat proteins were found associated with the host cell nuclear superstructure. We concluding that at early times after infection the viral templates mimic the state of the host cell chromatin by attaching to the cellular nuclear matrix.     

The ultrastructure of septal pores and associated structures in the ascogenous hyphae and asci ofSordaria humana

Summary Septal pores and associated structures have been studied in ascogenous hyphae, croziers and asci ofSordaria humana by means of electron microscopy of serial and random sections. Pores exhibit variable structures from relatively simple pore caps to complex swollen rims with associated membrane cisternae. The simple types are found at the base of the ascogenous hyphae while the complex forms occur at the apex, in the croziers and in very young, presporulation asci. Post-sporulation asci contain a relatively simple type of pore structure. Cells which subtend the ascogenous hyphae exhibit both open and capped pores in their cross walls. Pore structures may be asymmetric in which case they show greater complexity on the side of the cross wall nearest to the apex or crozier. Membranous components of the complex pores are continuous with the endomembrane systems of the two adjacent cells and thus with the outer membranes of the nuclear envelopes. Membrane continuities may connect prefusion nuclei or fusion nuclei in penultimate cells, with nuclei of the stalk and terminal cells of croziers. Some speculation is presented as to the implication and possible roles of these structures in relation to cell differentiation within the ascogenous hyphae and croziers.     

Propyl gallate,a free radical scavenger,counteracts the benefits of exogenously applied salicylic acid and aggravates the deleterious effects of the southern bean mosaic virus in Rhizobium-nodulated Phaseolus vulgaris plants

Infection of Rhizobium-nodulated Phaseolus vulgaris by the southern bean mosaic virus (SBMV) markedly decreased the growth and nodulation of plants. Exogenous applications of salicylic acid (SA) at concentrations ≥10 μM further decreased growth and nodulation of virus-infected (V) plants. Only SA concentration of 5 μM in the solution improved the growth, nodulation, chlorophyll concentration and the catabolism of ureide in leaves of V plants. The spray of leaves with 2 mM propyl gallate (+Pg) decreased growth, nodulation as well as the chlorophyll and leaf ureide concentrations in V plants, regardless of the concentration of SA at which plants were grown. Ultrastructural damages in leaf cells caused by SBMV were also enhanced in V+Pg plants. The massive proliferation of virus particles and the presence of virus crystalline arrays within symbiosomes of nodules in V+Pg plants were attributed to proliferation of branched plasmodesmata in leaf vascular-parenchyma cell walls that facilitated virus movement. Virus particles were never observed in leaf and nodule tissues of V plants not sprayed with Pg. Exogenous applications of SA hindered while Pg increased the symbiotic performance of H plants, pointing out the complexity to be addressed in breeding for virus resistance in Rhizobium-nodulated beans.     

Clostridium botulinum C2 toxin: low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells

The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADP-ribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.     

Reconstitution of herpes simplex virus type 1 nuclear capsid egress in vitro

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.     

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.     

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.     

Cell-to-cell transport via the lumen of the endoplasmic reticulum

Plasmodesmata are plasma membrane‐lined channels through which cytoplasmic molecules move from cell‐to‐cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3‐kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4‐kDa recombinant ER‐lumenal reporter protein (LRP) from a fragment of the endogenous ER‐lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER‐GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER‐lumenal molecules between cells.     

Noninvasive Monitoring of Apoptosis versus Necrosis in a Neuroblastoma Cell Line Expressing a Nuclear Pore Protein Tagged with the Green Fluorescent Protein

A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μMp-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.     

Ion channels in murine nuclei during early development and in fully differentiated adult cells

Summary The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.     

Changes in distribution of nuclear pores during differentiation of the male germ cells.

Changes in number of nuclear pores in different states of physiologica activity have been reported, but little is known about changing patterns of distribution in the course of cell differentiation. Pore distribution in male germ cells was studied in freeze fracture preparations of immature and mature rodent testis. As in other somatic cells, pores were uniformly and apparently randomly distributed in Sertoli cell nuclei. The nucleus of gonocytes and spermatogonia showed varying degrees of pore clustering. Spermatocytes invariably exhibited very striking pore aggregation with close hexagonal packing in pore-rich areas, and large pore-free areas. In early spermatids, pores appeared to be randomly distributed. As the acrosome formed and spread over the apical pole of the nucleus, pores disappeared ahead of its advancing margin and became more concentrated in the post-acrosomal region. The relationship of pore complexes to the chromosomes and the role of the fibrous lamina are discussed. The question as to whether the changing patterns observed involve movement of pores within fluid nuclear membranes, or a dissolution and reformation of new pores remains unanswered.