Lytic State Research Articles

Latent Herpesvirus Infection Augments Experimental Pulmonary Fibrosis

No effective treatment exists for idiopathic pulmonary fibrosis, and its pathogenesis remains unclear. Accumulating evidence implicates herpesviruses as cofactors (either initiating or exacerbating agents) of fibrotic lung disease, but a role for latent herpesvirus infection has not been studied. To develop a murine model to determine whether latent herpesvirus infection can augment fibrotic responses and to gain insight into potential mechanisms of enhanced fibrogenesis. Mice were infected with murine gammaherpesvirus 14 to 70 days before a fibrotic challenge with fluorescein isothiocyanate or bleomycin so that the virus was latent at the time of fibrotic challenge. Measurements were made after viral infection alone or after the establishment of fibrosis. gammaHerpesvirus is latent by 14 days post infection, and infection 14 to 70 days before fibrotic challenge augmented fibrosis. Fibrotic augmentation was not dependent on reactivation of the latent virus to a lytic state. Total cell numbers and fibrocyte numbers were increased in the lungs of latently infected mice administered fibrotic challenge compared with mock-infected mice that received fibrotic challenge. Latent infection up-regulates expression of proinflammatory chemokines, transforming growth factor-beta1, and cysteinyl leukotrienes in alveolar epithelial cells. Latent gammaherpesvirus infection augments subsequent fibrotic responses in mice. Enhanced fibrosis is associated with the induction of profibrotic factors and the recruitment of fibrocytes. Our data complement existing human and animal data supporting the hypothesis that gammaherpesviruses can serve as initiating cofactors in the pathogenesis of pulmonary fibrosis.

Why Do Phage Play Dice?

Phage lambda is among the simplest organisms that make a developmental decision. An infected bacterium goes either into the lytic state, where the phage particles rapidly replicate and eventually lyse the cell, or into a lysogenic state, where the phage goes dormant and replicates along with the cell. Experimental observations by P. Kourilsky are consistent with a single phage infection deterministically choosing lysis and double infection resulting in a stochastic choice. We argue that the phage are playing a "game" of minimizing the chance of extinction and that the shift from determinism to stochasticity is due to a shift from a single-player to a multiplayer game. Crucial to the argument is the clonal identity of the phage.

Kinetic Model of the Lysogeny/Lysis Switch of Phage λ

A kinetic model of the interactions between operators and regulators is developed to study the stabilities of genetic states and lysogeny/lysis switch in Escherichia coli infected by bacteriophage lambda. Using adiabatic approximation, the kinetic evolutions of mRNA and regulator concentrations can be deduced from operators' equations. Furthermore, the stability of each state of the system is studied. The results show that the lysogenic state switches to the lytic state through two bifurcations: one from a single stable state to a three-point state, and the other from a three-point state to a single stable state. Then we indicate that the property of the lysogeny/lysis switch satisfies the topological characteristics theorem. Finally, the influence of the left operators on the lysogeny/lysis switch is briefly discussed. The results show that the cooperativity of the CI2 bound to left and right operators makes the lysogenic state more stable.

Biologists who count

Biologists who count

Activation of the ERK signal transduction pathway by Epstein–Barr virus immediate-early protein Rta

BRCA1-associated protein 2 (BRAP2) is known to interact with the kinase suppressor of Ras 1 (KSR1), inhibiting the ERK signal transduction cascade. This study found that an Epstein-Barr virus (EBV) immediate-early protein, Rta, is a binding partner of BRAP2 in yeast and confirmed the binding in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation in 293(maxi-EBV) cells. Binding studies also showed that Rta and KSR1 interacted with the C-terminal 202 aa region in BRAP2. Additionally, Rta appeared to prevent the binding of KSR1 to BRAP2, activating the ERK signal transduction pathway and the transcription of an EBV immediate-early gene, BZLF1. Activation of the ERK signal transduction pathway by Rta may be critical for the maintenance of the lytic state of EBV.

Nuclear Factor-Y and Epstein Barr Virus in Nasopharyngeal Cancer

The Epstein Barr virus (EBV) is intimately associated with nasopharyngeal cancer (NPC) in a latent state expressing a limited number of genes. The process of switching from latency to replication is not well understood, particularly in response to DNA stress; hence, the focus of this study is on an EBV-positive NPC model. C666-1 cells were exposed to radiation (2-15 Gy) or cisplatin (0.1-50 microg/mL) assayed subsequently for relative EBV copy number (BamHI) and lytic gene expression (BRLF1 and BZLF1) using quantitative real-time PCR. Chromatin immunoprecipitation was conducted to assess the interaction of the transcription factor nuclear factor-Y (NF-Y) with promoter sequences. Radiation-induced and cisplatin-induced BamHI expression, along with increased levels of BRLF1 and BZLF1 in a dose-dependent and time-dependent manner, associated with the immediate nuclear transactivation of the transcription factor NF-Y and its own increased transcription of NF-Y subunits 8 h posttreatment. In silico analysis revealed three putative NF-Y consensus-binding sequences in the promoter region of BRLF1, which all interacted with NF-Y in response to radiation and cisplatin, confirmed using chromatin immunoprecipitation. Introduction of dominant-negative NF-YA reduced BRLF1 expression after radiation and cisplatin by 2.8-fold; in turn, overexpression of NF-YA resulted in a 2-fold increase in both BRLF1 and BZLF1 expression. These results show that NF-Y is an important mediator of EBV stress response in switching from a latent to lytic state. This novel insight could provide a potential therapeutic strategy to enhance NPC response to radiation and cisplatin.

Alfimeprase: a novel recombinant direct-acting fibrinolytic

Alfimeprase is a recombinant, direct-acting fibrinolytic zinc metalloprotease. Alfimeprase has direct proteolytic activity primarily against the fibrin(ogen) Aα chain. Alfimeprase is covalently bound and neutralised by serum α2-macroglobulin, a prevalent mammalian protease inhibitor. Preclinical pharmacology studies have shown that fibrinolysis with alfimeprase is up to sixfold more rapid than with select plasminogen activators, such as tissue-type plasminogen activator and urokinase. Alfimeprase directly delivered to a site of thrombosis has the potential to be a fast and effective fibrinolytic, which does not generate the systemic lytic state seen with plasminogen activators that is associated with major bleeding, including intracerebral haemorrhage. Phase I and II studies in individuals with arterial or venous thrombotic events indicate that alfimeprase is active and generally well tolerated.

A new look at bacteriophage lambda genetic networks.

Bacteriophage λ is a temperate bacteriophage, meaning that it can reproduce and develop either in a lytic or lysogenic state. When λ infects its bacterial host Escherichia coli , the phage may develop lytically, causing cell lysis with the release of hundreds of progeny virus, or it may abort

Incorporation of Fragment X into Fibrin Clots Renders Them More Susceptible to Lysis by Plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.

Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach.

Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. It has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cI, because lac repressor could not repress P(RM) in our constructs. Repression of cI by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.

Positive autoregulation of cI is a dispensable feature of the phage lambda gene regulatory circuitry.

Complex gene regulatory circuits contain many features that are likely to contribute to their operation. It is unclear, however, whether all these features are necessary for proper circuit behavior or whether certain ones are refinements that make the circuit work better but are dispensable for qualitatively normal behavior. We have addressed this question using the phage lambda regulatory circuit, which can persist in two stable states, the lytic state and the lysogenic state. In the lysogenic state, the CI repressor positively regulates its own expression by stimulating transcription from the P(RM) promoter. We tested whether this feature is an essential part of the regulatory circuitry. Several phages with a cI mutation preventing positive autoregulation and an up mutation in the P(RM) promoter showed near-normal behavior. We conclude that positive autoregulation is not necessary for proper operation of the lambda circuitry and speculate that it serves a partially redundant function of stabilizing a bistable circuit, a form of redundancy we term "circuit-level redundancy." We discuss our findings in the context of a two-stage model for evolution and elaboration of regulatory circuits from simpler to more complex forms.

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage λ Q − vector

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

Why the Lysogenic State of Phage λ Is So Stable: A Mathematical Modeling Approach

We develop a mathematical model of the phage λ lysis/lysogeny switch, taking into account recent experimental evidence demonstrating enhanced cooperativity between the left and right operator regions. Model parameters are estimated from available experimental data. The model is shown to have a single stable steady state for these estimated parameter values, and this steady state corresponds to the lysogenic state. When the CI degradation rate ( γ cI) is slightly increased from its normal value ( γ cI ≃ 0.0 min −1), two additional steady states appear (through a saddle-node bifurcation) in addition to the lysogenic state. One of these new steady states is stable and corresponds to the lytic state. The other steady state is an (unstable) saddle node. The coexistence these two globally stable steady states (the lytic and lysogenic states) is maintained with further increases of γ cI until γ cI ≃ 0.35 min −1, when the lysogenic steady state and the saddle node collide and vanish (through a reverse saddle node bifurcation) leaving only the lytic state surviving. These results allow us to understand the high degree of stability of the lysogenic state because, normally, it is the only steady state. Further implications of these results for the stability of the phage λ switch are discussed, as well as possible experimental tests of the model.

Disruption of Epstein-Barr virus latency in the absence of phosphorylation of ZEBRA by protein kinase C.

ZEBRA protein converts Epstein-Barr virus (EBV) infection from the latent to the lytic state. The ability of ZEBRA to activate this switch is strictly dependent on the presence of serine or threonine at residue 186 of the protein (A. Francis, T. Ragoczy, L. Gradoville, A. El-Guindy, and G. Miller, J. Virol. 72:4543-4551, 1999). We investigated whether phosphorylation of ZEBRA protein at this site by a serine-threonine protein kinase was required for activation of an early lytic cycle viral gene, BMRF1, as a marker of disruption of latency. Previous studies suggested that phosphorylation of ZEBRA at S186 by protein kinase C (PKC) activated the protein (M. Baumann, H. Mischak, S. Dammeier, W. Kolch, O. Gires, D. Pich, R. Zeidler, H. J. Delecluse, and W. Hammerschmidt, J. Virol 72:8105-8114, 1998). Two residues of ZEBRA, T159 and S186, which fit the consensus for phosphorylation by PKC, were phosphorylated in vitro by this enzyme. Several isoforms of PKC (alpha, beta(1), beta(2), gamma, delta, and epsilon ) phosphorylated ZEBRA. All isoforms that phosphorylated ZEBRA in vitro were blocked by bisindolylmaleimide I, a specific inhibitor of PKC. Studies in cell culture showed that phosphorylation of T159 was not required for disruption of latency in vivo, since the T159A mutant was fully functional. Moreover, the PKC inhibitor did not block the ability of ZEBRA expressed from a transfected plasmid to activate the BMRF1 downstream gene. Of greatest importance, in vivo labeling with [(32)P]orthophosphate showed that the tryptic phosphopeptide maps of wild-type ZEBRA, Z(S186A), and the double mutant Z(T159A/S186A) were identical. Although ZEBRA is a potential target for PKC, in the absence of PKC agonists, ZEBRA is not constitutively phosphorylated in vivo by PKC at T159 or S186. Phosphorylation of ZEBRA by PKC is not essential for the protein to disrupt EBV latency.

Epstein-Barr Virus BZLF1 Gene Is Activated by Transforming Growth Factor-β through Cooperativity of Smads and c-Jun/c-Fos Proteins

Induction of Epstein-Barr virus (EBV) production in an EBV-positive cell is achieved by expression of the gene BZLF1 that switches the latent state into a lytic state. The expression of the BZLF1 gene is initiated from the promoter Zp, which is normally suppressed in EBV-transformed B cells. The BZLF1 gene can be induced for expression by activating agents, such as transforming growth factor-beta (TGF-beta) and 12-O-tetradecanoylphorbol-13-acetate. The 12-O-tetradecanoylphorbol-13-acetate-responsive element located in the Zp is the AP-1 motif. The TGF-beta-responsive element, however, has not been determined. We demonstrated that the Smad4-binding element site, GTCTG, from -233 to -229, was located in the regulatory region of the Zp relative to the BZLF1 transcription initiation site and was physically associated with Smad4. This association was important for the TGF-beta induction of Zp. We also showed from the results of co-transfection experiments and electrophoretic mobility shift assays that both the AP-1 motif and Smad4-binding element site appeared to be required for the TGF-beta-induced activation of Zp. This effect was mediated through the cooperation of Smad3/Smad4 and c-Jun/c-Fos that formed a complex. TGF-beta treatment of Rael cells induced production of infectious EBV particles that was capable of infecting EBV-negative CA46 cells and transforming normal cord blood B cells, in vitro. Those data support a mechanism that TGF-beta induces the latent EBV in cells to enter the viral lytic cycle through regulation of key viral proteins by TGF-beta signal transducers. Those findings also suggest a role of TGF-beta in EBV-associated diseases.

Excimer Laser Revascularisation: Current Indications, Applications and Techniques

The ultraviolet pulsed excimer laser (308 nm wavelength) is currently the only laser approved by the FDA for percutaneous intervention in patients with ischemic coronary artery disease. The clinical presentation of the treated patients varies from stable and unstable angina to acute myocardial infarction. Potential advantages of excimer laser revascularisation in acute coronary syndromes and in ischaemic obstructive peripheral vascular disease include concomitant plaque debulking and thrombus removal; absence of systemic lytic state; shortened thrombus clearing time and facilitation of adjunct balloon angioplasty and stenting. Improved understanding of laser-tissue interactions and positive clinical outcomes through the use of safe lasing techniques have led to expansion of indications/applications for laser angioplasty. These include stent restenosis, complex lesions and thrombotic stenoses, bifurcation lesions, balloon failure, total occlusions, focal saphenous vein graft lesions and peripheral arterial obstructions. The excimer laser can be effectively utilised in patients with depressed left ventricular ejection fraction and does not require implantation of a temporary pacemaker as no-reflow phenomenon and severe arrhythmias are rarely encountered. Careful case selection, proper utilisation of equipment and incorporation of efficient lasing techniques play a crucial role in effective and safe cardiovascular laser applications.

Analysis of two-stage continuous operation of Escherichia coli containing bacteriophage λ vector

Bacteriophage λ containing a cloned-gene is stably maintained in Escherichia coli in the lysogenic state while it is replicated and it overproduces a recombinant protein product in the lytic state. The host cell is eventually lysed in the lytic state. The kinetics of cell lysis and production induction were studied and are reported in this article through model equations. In two-stage continuous operation, the first tank is maintained in the lysogenic state for cell growth and cloned-gene stability while the second tank is in the lytic state for the overproduction of cloned-gene product. Individual cells in the second tank have different extent of the induction for product formation, since each has a different residence time. The different residence time for individual cells was taken into account using a population model. The numerical results show good agreement with the experimental data for the prediction of dilution rate in the second tank which gives the maximum product concentration.

Optimization of bacteriophage λQ −-containing recombinant Escherichia coli fermentation process

Q− mutant phage λ is deficient in the synthesis of the proteins involved in cell lysis and λ DNA packaging. As a result, the replicated Q−λ DNA containing a cloned gene is not easily coated by a phage head and remains naked for the ample expression of the cloned gene, and also the host cells do not lyse easily and larger amounts of cloned gene products are produced. In a two-phase operation, the first phase is operated at a low temperature to keep the phage in the lysogenic state for cell growth and cloned gene stability, while the second phase is operated at a high temperature to induce the lytic state for the amplification of the cloned gene and overproduction of its product. This two-phase operation was optimized by determining both the optimal temperatures for the growth and production phases and the optimal switching time between the growth to the production phase. The optimal temperatures for growth and production phases were 33 and 40 °C, respectively. The optimal switching time was 3 h. The recombinant β-galactosidase production using this optimal process was about 20 times higher than in the single-copy lysogenic state.

Requirement for cell-to-cell contact in Epstein-Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes.

Two nasopharyngeal carcinoma (NPC) cell lines and one keratinocyte cell line could be infected with Epstein-Barr virus (EBV) by cocultivation with virus-producing cells but not by cell-free virus. Using porous culture inserts to manipulate the cell-to-cell contact, we demonstrated that contact between EBV donor B cells and EBV recipient epithelial cells was required for the infection. Cell-to-cell contact not only provided a CR2-independent route of infection but also enhanced CR2-mediated infection in a synergistic manner. Activity of two EBV promoters (Cp and Wp) and expression of EBNA2 were detected in the infected population. A small proportion of the infected cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical treatment. This study provides information on how EBV may infect epithelial cells in vivo, such as at the onset of NPC development.

The biology of Epstein-Barr virus in post-transplant lymphoproliferative disease.

Post-transplant lymphoproliferative disease (PTLD) is a B cell proliferative disorder that is associated with Epstein-Barr virus (EBV), an ubiquitous herpesvirus. EBV-seronegative organ transplant recipients are at highest risk. EBV infection in PTLD lesions exists in a latent rather than lytic state, making tumor regression in response to antiviral agents unlikely. Viral latency proteins drive proliferation of T cells but also allow T cells to target PTLD lesions for destruction. Augmentation of the cellular immune response via the infusion of EBV-specific cytotoxic T cells has yielded promising results in the prevention and treatment of PTLD in bone marrow transplant recipients. Efforts to extend this strategy to solid organ transplant recipients are ongoing.