Traumatic oil glands induced by pruning in the wound-associated phloem of Eucalyptus globulus: chemistry and histology

The natural occurrence of oil glands in various organs such as bark and leaves is well established as a characteristic of Eucalyptus, but this is the first reported case of traumatic oil glands induced in response to wounding. The new phloem enveloping the wound, which had developed within the 2 years following branch pruning in 5-year-old Eucalyptus globulus Labill., was morphologically distinct from healthy stem phloem. Histological examinations revealed this wound-associated phloem to be largely composed of secretory cavities similar in appearance to oil glands. Subsequent analysis of the wound-associated phloem extracts by GC-MS confirmed the presence of volatile terpenes and phenols. The total extracted oil content determined for wound-associated phloem extracts was significantly higher (>4 times) than for healthy stem phloem extracts. A comparison of the relative abundances of ten individual terpenoids from wound-associated phloem and healthy phloem revealed a number of significant differences in terpene composition. Implications of the role of terpenes as inducible secondary metabolites in tree wound responses are discussed.     

Charged residues in hepatitis C virus NS4B are critical for multiple NS4B functions in RNA replication

The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N- and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex.     

Junctional communication is induced in migrating capillary endothelial cells

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.     

A Plasma Membrane Wound Proteome: REVERSIBLE EXTERNALIZATION OF INTRACELLULAR PROTEINS FOLLOWING REPARABLE MECHANICAL DAMAGE

Cells in mechanically active tissues undergo constant plasma membrane damage that must be repaired to allow survival. To identify wound-associated proteins, a cell-impermeant, thiol-reactive biotinylation reagent was used to label and subsequently isolate intracellular proteins that become exposed on the surface of cultured cells after plasma membrane damage induced by scraping from substratum or crushing with glass beads. Scrape-damaged cells survived injury and were capable of forming viable colonies. Proteins that were exposed to the cell surface were degraded or internalized a few seconds to several minutes after damage, except for vimentin, which was detectable on the cell surface for at least an hour after injury. Seven major biotinylated protein bands were identified on SDS-PAGE gels. Mass spectrometric studies identified cytoskeletal proteins (caldesmon-1 and vimentin), endoplasmic reticulum proteins (ERp57, ERp5, and HSP47), and nuclear proteins (lamin C, heterogeneous nuclear ribonucleoprotein F, and nucleophosmin-1) as major proteins exposed after injury. Although caldesmon was a major wound-associated protein in calpain small subunit knock-out fibroblasts, it was rapidly degraded in wild-type cells, probably by calpains. Lamin C exposure after wounding was most likely the consequence of nuclear envelope damage. These studies document major intracellular proteins associated with the cell surface of reversibly damaged somatic cells. The studies also show that externalization of some proteins reported to have physiologic or pathologic roles on the cell surface can occur in cells undergoing plasma membrane damage and subsequent repair.     

Wound-Associated Competency Factors Are Required for the Proximal Cell Responses of Soybean to the Phytophthora sojae Wall Glucan Elicitor

Intact soybean (Glycine max L. [Merr.]) tissues show distinct proximal and distal cell responses to the Phytophthora sojae (Kauf. and Gerde.) wall glucan elicitor. Proximal cells respond with accumulations of glyceollin and phenolic polymers, whereas distal cells respond with an increase of isoflavone conjugates. Comparison of the activities of the P. sojae glucan in the classical cut cotyledon and a cotyledon infiltration assay suggests that the proximal, but not the distal, responses to elicitor require tissue wounding. Washing the surface of cut cotyledons prior to elicitor treatment also greatly diminishes the proximal responses, which can be restored in a dose-dependent manner by prior treatment of the washed cells with wound exudate from cut "donor" cotyledons. Thus, discrete wound-associated factors, which we term elicitation competency factors, are required for the proximal cell response to the glucan elicitor. The wound factors induce a competent state that is transient in nature. Maximal elicitor response is seen 2 to 3 h after wounding, and cells become elicitor nonresponsive after 4 h. Competency is markedly affected by the age of tissues; cotyledons become more inherently competent as they approach senescence. The time course of attainment of the competent state and its duration are strongly affected by light and temperature. Since the wound-associated competency factors can also be obtained from washings of hypersensitive lesions, we hypothesize that similar competency factors may be released from hypersensitively dying cells in incompatible infections. This event may program the immediately surrounding cells to make them competent for the proximal defense responses.     

Allelic variation in the hepatitis C virus NS4B protein dramatically influences RNA replication

In the Huh-7.5 hepatoma cell line, replication of the genotype 1a H77 strain of hepatitis C virus (HCV) is attenuated compared to that of the genotype 1b Con1 strain. This study identifies the poorly characterized integral membrane protein, NS4B, as a major determinant for this replication difference. Chimeric H77 subgenomic replicons containing the entire NS4B gene from Con1 in place of the H77 NS4B sequence replicated approximately 10-fold better than the H77 parent and to levels similar to that of the adapted Con1 replicon. An intermediate level of replication enhancement was conferred by H77 chimeras containing the poorly conserved N-terminal 47 residues or the remaining less-divergent C terminus of Con1 NS4B. The replication-enhancing activity within the N terminus of NS4B was further mapped to two Con1-specific amino acids. Experiments to elucidate the mechanism of enhanced H77 replication revealed that Con1 NS4B primarily increased H77 RNA synthesis on a per cell basis, as indicated by the similar capacities of chimeric and parental replicons to establish replication in Huh-7.5 cells and the higher levels of both positive- and negative-strand RNAs for the chimeras than for the H77 parent. Additionally, enhanced H77 replication was not the result of Con1 NS4B-mediated effects on HCV translation efficiency or alterations in polyprotein processing. Expression of Con1 NS4B in trans did not improve the replication of the H77 parental replicon, suggesting a cis-dominant role for NS4B in HCV replication. These results provide the first evidence that allelic variation in the NS4B sequence between closely related isolates significantly impacts HCV replication in cell culture.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Interaction of Bartonella henselae with endothelial cells results in rapid bacterial rRNA synthesis and replication

Bartonella henselae is a slow-growing microorganism and the causative pathogen of bacillary angiomatosis in man. Here, we analysed how interaction of B. henselae with endothelial cells might affect bacterial growth. For this purpose, bacterial rRNA production and ribosome content was determined by fluorescence in situ hybridization (FISH) using rRNA-targeted fluorescence-labelled oligonucleotide probes. B. henselae grown on agar plates showed no detectable rRNA content by means of FISH, whereas B. henselae co-cultured with endothelial cells showed a rapid increase of rRNA production within the first 18 h after inoculation. The increased rRNA synthesis was paralleled by a ∼1000-fold intracellular bacterial replication, whereas bacteria grown on agar base showed only a ∼10-fold replication within the first 48 h of culture. Pretreatment of host cells with paraformaldehyde prevented adhesion, invasion, intracellular replication and bacterial rRNA synthesis of B. henselae . In contrast, inhibition of host cell protein synthesis by cycloheximide did not affect bacterial adhesion and invasion, but prevented intracellular replication although bacterial rRNA content was increased. Inhibition of actin polymerization by cytochalasin D did not affect adhesion, invasion, increased rRNA content or intracellular replication of B. henselae. These results demonstrate that rRNA synthesis and replication of B. henselae is promoted by viable host cells with intact de novo protein synthesis.     

NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Inhibition of pseudorabies virus replication by vesicular stomicles virus I. Activity of infectious and inactivated B particles.

Infectious B particles of vesicular stomatitis virus (VSV) are capable of inhibiting the replication of pseudorabies virus (PSR) in a variety of cell lines. Even under conditions of an abortive infection in a continuous line of rabbit cornea cells (RC-6O), B particles interfere with the replication of PSR with high efficiency. Particle per cell dose-response analysis of B particle populations revealed that the number of VSV particles capable of inhibiting PSR replication exceeds the number of PFU by a factor of 32 to 64. When B particles are treated with UV irradiation, a drastic increase in the multiplicity of infection is required to inhibit PSR replication. Whereas one infective B particles per cell is sufficient to prevent replication of PSR, 800 to 1,000 VSV particles rendered noninfective by UV irradiation are required to compensate for the loss of VSV synthetic activity that results from irradiation. Temperature-sensitive mutants representing five complementation groups of VSV were tested at low multiplicities of infection for their effect on PSR replication at the nonpermissive temperature. Generally, the ability of the different complementation groups to amplify virion products at the nonpermissive temperature is associated with their ability to inhibit PSR replication. These results imply that at low multiplicities of infection, amplification of infecting VSV components is necessary for inhibition of PSR replication., but at high multiplicities of infection with VSV, a virion component can prevent PSR replication in the absence of de novo VSV RNA or protein synthesis.     

Multiple cellular factors bind to cis-regulatory elements found inboard of the 5' palindrome of minute virus of mice.

Previous genetic analysis of the DNA replication of minute virus of mice (MVM) minigenomes suggested that specific elements, A (nucleotides [nt] 4489 to 4636) and B (nt 4636 to 4695), found inboard of the 5' palindrome are required for efficient MVM DNA replication (P. Tam and C. R. Astell, Virology 193:812-824, 1993). In this report, we show that two MVM RsaI restriction fragments (RsaI A [nt 4431 to 4579] and RsaI B [nt 4579 to 4662]) are able to activate DNA replication of an MVM minigenome containing deletions of both elements A and B. We also show that sequences inboard of the right palindrome are able to activate replication of minigenomes containing two left termini. In order to investigate the importance of the RsaI fragments, we demonstrate the presence of a number of sequence-specific DNA-protein interactions by electrophoretic mobility shift assays. After partial fractionation of A9 nuclear extracts, DNase I footprinting analysis was used to determine the binding sites for MVM replication factor (MRF) B5. MRF B5 protects two distinct regions (sites I and II) of the RsaI B probe from DNase I digestion. Competition f electrophoretic mobility shift assays with synthetic oligonucleotides corresponding to sites I and II suggest that MRF B5 is composed of two factors, MRF B3 and MRF B4, which bind DNA independently in a sequence-specific manner. It may be possible that these replication factors are proteins which are able to transactivate MVM DNA replication and hence are accessory replication factors.     

Human herpesvirus 6B origin: sequence diversity, requirement for two binding sites for origin-binding protein, and enhanced replication from origin multimers.

A previously identified human herpesvirus 6B (HHV-6B) origin of DNA replication contains two binding sites for the origin-binding protein (OBPH6B). We have investigated the functional significance of these sites by determining the replication efficiencies of mutated origin sequences, using a transient replication assay. The results indicate that both sites are required for DNA replication. In addition, we have tested the functional consequences of linear sequence amplifications in the origin. The data show that tandemized origin elements are more efficiently replicated than single-copy origins. Finally, we have determined the extent of interstrain origin sequence variation that exists among HHV-6 isolates by cloning, sequencing, and analyzing origins from a number of virus isolates, including examples of both HHV-6A and HHV-6B.     

A genetic interaction between hepatitis C virus NS4B and NS3 is important for RNA replication

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.