Wolbachia replication and host cell division in Aedes albopictus

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachiarsquos replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (M phi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to M phi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca2+/Mg2+ enhanced DV binding. This argues against involvement of beta (2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains. (C) 2001 Elsevier Science B.V. All rights reserved.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Transcriptome analysis of a cnidarian-dinoflagellate mutualism reveals complex modulation of host gene expression

Background: Cnidarian - dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian - algal symbiosis. Results: We detected statistically significant differences in host gene expression profiles between sea anemones ( Anthopleura elegantissima) in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress. Conclusion: Our data do not support the existence of symbiosis- specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian - dinoflagellate associations provides critical insights into the maintenance and regulation of the symbiosis.

Ovary colonization by Claviceps africana is related to ergot resistance in male-sterile sorghum lines

Ergot, caused by Claviceps africana, has emerged as a serious threat to sorghum hybrid seed production worldwide. In the absence of gene-for-gene-based qualitative resistance in commercial cultivars, varieties with high pollen production that can escape ergot infection are preferred. Recent demonstration of differences in ergot susceptibility among male-sterile lines has indicated the presence of partial resistance. Using chitin-specific fluorescin-isothiocyanate-conjugated wheat germ agglutin and callose-specific aniline blue, this study investigated the process of sorghum ovary colonization by C. africana. Conidia germinated within 24 h after inoculation (a.i.); the pathogen was established in the ovary by 79 h a.i., and at least half of the ovary was converted into sphacelial tissue by 120 h a.i. Changes in fungal cell wall chitin content and strategic callose deposition in the host tissue were associated with penetration and invasion of the ovary. The rate of ovary colonization differed in three male-sterile lines that also differed in ergot susceptibility. This work demonstrates a possible histological basis for partial resistance in male-sterile sorghum lines that could lay the foundation for variety improvement through further breeding and selection.

Evolution of Wolbachia pipientis transmission dynamics in insects

Wolbachia pipientis is an intracellular bacterial parasite of arthropods that enhances its transmission by manipulating host reproduction, most commonly by inducing cytoplasmic incompatibility. The discovery of isolates with modified cytoplasmic incompatibility phenotypes and others with novel virulence properties is an indication of the potential breadth of evolutionary strategies employed by Wolbachia.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellular viral components, and the heterologous product titers. The model describes the whole processes of viral infection and the effect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave good agreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infection in batch and fed-batch culture were studied using the model.

Role of LMP1 in immune control of EBV infection

The Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include up-regulation of expression of B cell activation antigens, adhesion molecules and various components of the antigen processing pathway. Here we discuss how LMP1 acts like an expression 'switch' which, depending on the stage of EBV infection, manoeuvres various pathways that either modulate the immune system towards or against its survival.

Mechanistic aspects of HIV-1 reverse transcription initiation

During reverse transcription, the positive-strand HIV-1 RNA genome is converted into a double-stranded DNA copy which can be permanently integrated into the host cell genome. Recent analyses show that HIV-1 reverse transcription is a highly regulated process. The initiation reaction can be distinguished from a subsequent elongation reaction carried out by a reverse transcription complex composed of (at least) heterodimeric reverse transcriptase, cellular tRNA(lys3) and HIV-1 genomic RNA sequences. In addition, viral factors including Tat, Nef, Vif, Vpr, IN and NCp7, cellular proteins, and TAR RNA and other RNA stem-loop structures appear to influence this complex and contribute to the efficiency of the initiation reaction. As viral resistance to many antiretroviral compounds is a continuing problem, understanding the ways in which these factors influence the reverse transcription complex will likely lead to novel antiretroviral strategies. Copyright (C) 2001 John Wiley Sons, Ltd.

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

Engineering mRNA translation initiation to enhance transient gene expression in Chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser51Ala site-directed mutant of eIF2, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2 by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single-and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2 Ser51Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2 protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2 phosphorylation in cells transfected with the mutant eIF2 construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser51Ala or wild-type eIF2 proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.

The EBNA- 3 gene family proteins disrupt the G2/M checkpoint

The Epstein - Barr nuclear antigens (EBNA), EBNA-3, -4 and - 6, have previously been shown to act as transcriptional regulators, however, this study identifies another function for these proteins, disruption of the G2/M checkpoint. Lymphoblastoid cell lines (LCLs) treated with a G2/M initiating drug azelaic bishydroxamine ( ABHA) did not show a G2/M checkpoint response, but rather they display an increase in cell death, a characteristic of sensitivity to the cytotoxic effects of the drug. Cell cycle analysis demonstrated that the individual expression of EBNA-3, - 4 or - 6 are capable of disrupting the G2/M checkpoint response induced by ABHA resulting in increased toxicity, whereas EBNA-2, and - 5 were not. EBNA-3 gene family protein expression also disrupted the G2/M checkpoint initiated in response to the genotoxin etoposide and the S phase inhibitor hydroxyurea. The G2 arrest in response to these drugs were sensitive to caffeine, suggesting that ATM/ATR signalling in these checkpoint responses may be blocked by the EBNA-3 family proteins. The function of EBNA-3, - 4 and - 6 proteins appears to be more complex than anticipated and these data suggest a role for these proteins in disrupting the host cell cycle machinery.