Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.     

Glucocorticoid receptor-induced down-regulation of MMP-9 by ginseng components, PD and PT contributes to inhibition of the invasive capacity of HT1080 human fibrosarcoma cells

We examined the effects of the purified ginseng components, panaxadiol (PD) and panaxatriol (PT), on the expression of matrix metalloproteinase-9 (MMP-9) in highly metastatic HT1080 human fibrosarcoma cell line. A significant down-regulation of MMP-9 by PD and PT was detected by Northern blot analysis. However, the expression of MMP-2 was not changed by treatment with PD and PT. Quantitative gelatin based zymography confirmed a markedly reduced expression of MMP-9, but not MMP-2 in the treatment of PD and PT. To investigate whether the reduced level of MMP-9 by PD and PT affects the invasive capacity of HT1080 cells, we conducted an in vitro invasion assay with PD and PT treated cells. The results of the in vitro invasion assay revealed that PD and PT reduced tumor cell invasion through a reconstituted basement membrane in the transwell chamber. Because of the similarity of chemical structure between PD, PT and dexamethasone (Dexa), a synthetic glucocorticoid, we investigated whether the down-regulation of MMP-9 by PD and PT were mediated by the nuclear translocation of glucocorticoid receptor (GR). Increased GR in the nucleus of HT1080 human fibrosarcoma cells treated by PD and PT was detected by immunocytochemistry. Western blot and gel retardation assays confirmed the increase of GR in the nucleus after treatment with PD and PT. These results suggest that GR-induced down-regulation of MMP-9 by PD and PT contributes to reduce the invasive capacity of HT1080 cells.     

NSP-interacting GTPase: A cytosolic protein as cofactor for nuclear shuttle proteins

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes.Key words: geminivirus, NIG, NSP, nucleocytoplasmic trafficking, transport activity     

Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review

Synthetic oligodeoxyribonucleotides (oligos) are now widely used as artificial regulators for gene expression both in cell-free media and in cultured cells. We describe the biological consequence of the various chemical modifications that have been introduced into the molecules to improve their resistance against nuclease attack, their affinity for the target mRNA and their uptake by cells. We also describe the rising generation of antimessenger oligos. Covalently linked to reactive groups these molecules direct irreversible modifications of the complementary nucleic acids. We anticipate that these oligos will be targeted to double-stranded nucleic acids to interfere with gene expression at the DNA level.     

Cellular targets of functional and dysfunctional mutants of tobacco mosaic virus movement protein fused to green fluorescent protein

Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.     

Experimental protein malnutrition in squirrel monkeys: cytochemical interaction of nucleolus and cytoplasm in the anterior horn cells of the spinal cord of Saimiri sciureus.

Nuecleolo-cytoplasmic relationships have been studied using histochemical techniques in the spinal cord of healthy neonates compared with those born to mothers malnourished through most of the gestation period, and healthy and severely protein-malnourished young adult squirrel monkeys. Formaldehyde-fixed as well as fresh frozen tissues were used for histological study and for histochemical techniques to demonstrate lipids, carbohydrates, nucleic acids and enzymes of various metabolic cycles. Changes in the neuronal cytoplasm of the low protein animals appear to be related to impaired protein metabolism and energy transport. The nucleolus reacts to cytoplasmic changes by enlarging its size and by active synthesis of secretion of nucleolar material, which is then passed into the cytoplasm through an evagination of the nuclear envelope. The greater the degree of chromatolysis in the cytoplasm, the more active the nucleolus appears to be in terms of its enlarged size and transfer of secretion products into the cytoplasm. It is believed that the perinuclear accumulation of secretion products in the neuronal cytoplasm of the protein-deficient animals has its origin in the nucleolus. Such an accumulation in the severely malnourished animals appears to be a compensatory mechanism for the increased cytoplasmic catabolism and loss of ribonucleoprotein material in order to facilitate additional protein synthesis for cell survival.     

Inhibition of RNA nucleo-cytoplasmic translocation by anti-nucleus antibody

Anti-nucleus antibody was raised by immunizing rabbits with rat liver nuclei, and purified by affinity-column chromatography. When purified anti-nucleus IgG molecules were introduced into FL cells by the erythrocyte-ghost fusion method with HVJ (Sendai virus), release of RNA from the nucleus into the cytoplasm was inhibited in the presence of alpha-amanitin, but nuclear accumulation of 125I-labeled non-histone chromosomal protein from the cytoplasm was not inhibited. These findings suggest that the nucleo-cytoplasmic transport mechanisms of RNA and nuclear proteins are different. The molecular weight of the antigen of this antibody was determined to be about 55K by the immunoblotting technique.     

Guanine Metabolism in Carbon-Limited Euglena gracilis

SYNOPSIS. Guanine-8-³H is not a specific label for nucleic acids in Euglena gracilis (strain SM-L1, streptomycin-bleached), but is incorporated into all biochemical fractions. Autoradiographic results show that guanine is metabolized in, both the nucleus and the cytoplasm of these Eugelna , but nuclear metabolism of guanine occurs only at a very low level in carbon-limited cells. Further, carbon-limited Euglena incorporate less total guanine than do control Euglena in a comparable time period. However, the majority of label incorporated into the nucleic acid fraction of the carbon-limited cells is incorporated into RNA.     

A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells

A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.     

The dynamics of karyopherin-mediated nuclear transport.

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

FINE STRUCTURAL AND RADIOAUTOGRAPHIC OBSERVATIONS ON DENSE PERINUCLEAR CYTOPLASMIC MATERIAL IN TADPOLE OOCYTES

Dense fibrous material is first seen in association with mitochondria in tadpole oogonia but is most prominent in oocytes during the extended first meiotic prophase when it aggregates into dense bodies in the perinuclear cytoplasm. The origin of this material has been attributed to 350-A nuclear granules which form cytoplasmic streamers of fibrous material upon passage through nuclear pores. This has commonly been interpreted as the transfer of ribonucleoprotein to the cytoplasm for storage. However, cytochemical reactions for nucleic acids have indicated an absence of detectable RNA in this dense material, and the results of radioautographic studies with labeled uridine, thymidine, or actinomycin D argue against the presence of nucleic acids. When sites of incorporation of tritiated amino acids were radioautographically localized, an appreciable number of silver grains were present over the dense bodies. Uptake of certain amino acids occurs fairly promptly but the degree of labeling levels off after about 6 hr, suggesting a rapid turnover of the material in the dense bodies. Attention is drawn to the similarity of the dense bodies to structures present in germ cells of a number of other species, and possible functions of the dense bodies in germ cell differentiation are considered.     

Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.     

A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.     

ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus.     

Effects of brequinar and ciprofloxacin on de novo nucleotide biosynthesis in mouse L1210 leukemia

Exposure of mouse L1210 leukemia cells to 25 microM brequinar for 4 h results in large accumulations of N-carbamyl-L-aspartate and L-dihydroorotate to cellular concentrations of 8.5 mM and 0.8 mM, respectively, while UTP and CTP decrease to 4% of their initial levels; incorporation of [14C]bicarbonate into nucleic acids (DNA and RNA) was decreased to 47%. These data provide direct evidence for inhibition of DHO dehydrogenase by brequinar in growing cells. Exposure of leukemia cells to 200 microM ciprofloxacin for 4 h did not affect de novo pyrimidine nucleotide biosynthesis or the incorporation of [14C]bicarbonate into nucleic acids but resulted in a general decrease in nucleoside triphosphates, with concomitant accumulation of nucleoside mono- and diphosphates (the adenylate energy charge decreased from 0.89 to 0.69), consistent with inhibition of the electron transport chain or uncoupling of oxidative phosphorylation.     

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.