Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication.

Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine.     

Incorporation of S-[3H]methyl-d-cysteine into the peptidoglycan of ether-treated cells of Escherichia coli

Certain D-amino acids can be incorporated into the murein sacculus of Escherichia coli apparently through a direct transpeptidation reaction independent of the normal biosynthetic pathway. Investigation of this process is important because it could lead to the identification of hitherto unknown enzymes involved in murein metabolism. However, a serious drawback is the lack of an appropriate in vitro assay. We have analysed the suitability of a system based on the incorporation of a radioactive substrate (S-[3H]methyl-D-cysteine) by ether-treated cells, a method successfully applied before to the study of murein biosynthesis. The results reported here indicate that ether-treated cells are indeed proficient in the incorporation of D-amino acids, matching closely the properties of the reaction in growing cells.     

Mechanism of inhibition of herpes simplex virus and vaccinia virus DNA polymerases by aphidicolin, a highly specific inhibitor of DNA replication in eucaryotes.

The inhibition in vitro of herpes simplex virus 1 and vaccinia virus DNA polymerases by aphidicolin is primarily noncompetitive with dGTP, dATP, dTTP, DNA, and Mg2+ and competitive with dCTP in analogy with the mode of inhibition of cellular alpha-polymerase. The degree of inhibition of viral or cellular growth in vivo can be quantitatively predicted by the degree of inhibition of the isolated replicative DNA polymerases at the same concentration of aphidicolin in suitable conditions (limiting dCTP concentration). Thus, the only in vivo target for aphidicolin is probably the replicative DNA polymerase, and aphidicolin is a highly specific inhibitor of replicative nuclear DNA synthesis in eucaryotes. This, coupled with the lack of mutagenic effect, represents a valuable property for an anticancer drug. The specificity of inhibition (contrary to the aspecific effect on almost all DNA polymerases by a true competitive inhibitor, such as 1-beta-D-arabinofuranosylcytidine 5'-triphosphate) and the structure of the drug, which does not resemble that of the triphosphates, suggest that aphidicolin must recognize a site common only to the replicative DNA polymerases of eucaryotes and different from the binding site for deoxyribonucleic triphosphates and DNA, which should be similar in reparative and procaryote-type DNA polymerase; the aphidicolin binding site is probably very near to, or even overlaping with, the binding site for dCTP so that the drug mimics a competitive effect with this nucleotide.     

O-Acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis

O-Acetylation of the MurNAc moiety of peptidoglycan is typically associated with bacterial resistance to lysozyme, a muramidase that serves as a central component of innate immunity. Here, we report that the peptidoglycan of Bacillus anthracis, the etiological agent of anthrax, is O-acetylated and that, unusually, this modification is produced by two unrelated families of O-acetyltransferases. Also, in contrast to other bacteria, O-acetylation of B. anthracis peptidoglycan is combined with N-deacetylation to confer resistance of cells to lysozyme. Activity of the Pat O-acetyltransferases is required for the separation of the daughter cells following bacterial division and for anchoring of one of the major S-layer proteins. Our results indicate that peptidoglycan O-acetylation modulates endogenous muramidase activity affecting the cell-surface properties and morphology of this important pathogen.     

Bacillus subtilis CwlP of the SP-{beta} prophage has two novel peptidoglycan hydrolase domains, muramidase and cross-linkage digesting DD-endopeptidase

For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-β prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn(2+)-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn(2+) dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

Mechanistic effects of kijimicin on inhibition of human immunodeficiency virus replication

Kijimicin represents an important type of ionophore compound. In veterinary medicine, it is becoming important as anticoccidiostatic agent and feed supplement. We examined Kijimicin for its HIV inhibitory activity. The compound exhibited concentration-dependent inhibition of HIV replication in primary infected cultures of human T-lymphoblastoid H9 cells. Substantial inhibition of viral replication was observed at concentrations of Kijimicin that showed little cytotoxicity. The ratio of IC₅₀ values for the MTT to RT assays was 40. Anti HIV activity was also observed in cultures of monocytic lineage U937 cells chronically infected with HIV. Moreover, in attempting to define the inhibitory mechanism of Kijimicin, we investigated its effect on each step of HIV replication. The infectivity of progeny viral particles was reduced by Kijimicin treatment. This decrease may be due to incompletely glycosylated forms of gp120.     

RNA interference mediated inhibition of Chikungunya virus replication in mammalian cells

Chikungunya has emerged as one of the most important arboviral infection of public health significance. Recently several parts of Indian Ocean islands and India witnessed explosive, unprecedented epidemic. So far, there is no effective antiviral or licensed vaccine available against Chikungunya infection. RNA interference mediated inhibition of viral replication has emerged as a promising antiviral strategy. In this study, we examined the effectiveness of small interfering RNAs (siRNAs) against the inhibition of Chikungunya virus replication in Vero cells. Two siRNAs against the conserved regions of nsP3 and E1 genes of Chikungunya virus were designed. The siRNA activity was assessed by detecting both the infectious virus and its genome. The results indicated a reduction of virus titer up to 99.6% in siRNA transfected cells compared to control. The viral inhibition was most significant at 24 h (99%), followed by 48 h (65%) post infection. These results were also supported by the quantitative RT-PCR assay revealing similar reduction in Chikungunya viral genomic RNA. The siRNAs used had no effect on the expression of house keeping gene indicating non-interference in cellular mechanism. The specific and marked reduction in viral replication against rapidly replicating Chikungunya virus achieved in this study offers a potential new therapeutic approach. This is the first report demonstrating the effectiveness of siRNA against in vitro replication of Chikungunya virus.     

Stable inhibition of hepatitis B virus expression and replication by expressed siRNA

RNA interference might be an efficient antiviral therapy for some obstinate illness. Here, we studied the effects of hepatitis B virus (HBV)-specific 21-nt small interfering RNAs (siRNA) on HBV gene expression and replication in 2.2.15 cells. Seven vectors expressing specific hairpin siRNA driven by the RNA polymerase II-promoter were constructed and transfected into 2.2.15 cells. In the cell strain that can stably express functional siRNA, the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg) secretion into culture media was inhibited by 86% and 91%, respectively, as shown by an enzyme-linked immunosorbent assay. Immunofluorescence and Western blot indicated similar results. HBV DNA was markedly restrained by 3.28-fold, as assessed by the fluorescent quantitation PCR. Moreover, the HBV mRNA was significantly reduced by 80% based on semiquantitative RT-PCR. In conclusion, the specific siRNA can knock down the HBV gene expression and replication in vitro, and the silence effects have no relationship with interferon response.     

Structural determinants of caspase-9 inhibition by the vaccinia virus protein, F1L

In multicellular organisms, apoptosis is a powerful method of host defense against viral infection. Apoptosis is mediated by a cascade of caspase-family proteases that commit infected cells to a form of programmed cell death. Therefore, to replicate within host cells, viruses have developed various strategies to inhibit caspase activation. In the mitochondrial cell-death pathway, release of cytochrome c from mitochondria into the cytosol triggers assembly of the oligomeric apoptosome, resulting in dimerization and activation of the apical caspase-9 (C9), and in turn its downstream effector caspases, leading to apoptosis. We previously showed that the vaccinia virus-encoded Bcl-2-like protein, F1L, which suppresses cytochrome c release by binding Bcl-2 family proteins, is also a C9 inhibitor. Here, we identify a novel motif within the flexible N-terminal region of F1L that is necessary and sufficient for interaction with and inhibition of C9. Based on functional studies and mutagenesis, we developed an atomic model of the complex in which F1L inhibits C9 by engaging the active site in the reverse orientation with respect to substrate peptides, in a manner analogous to that of XIAP-mediated inhibition of caspases-3 and -7. These studies offer new insights into the mechanism of apoptosome inhibition by F1L as well as novel probes to understand the molecular bases of apoptosome regulation and turnover. They also suggest how the two distinct functionalities of F1L (inhibition of C9 and suppression of pro-apoptotic Bcl-2 family proteins) may operate in a cellular setting.     

Locked nucleic acid containing antisense oligonucleotides enhance inhibition of HIV-1 genome dimerization and inhibit virus replication

We have evaluated antisense design and efficacy of locked nucleic acid (LNA) and DNA oligonucleotide (ON) mix-mers targeting the conserved HIV-1 dimerization initiation site (DIS). LNA is a high affinity nucleotide analog, nuclease resistant and elicits minimal toxicity. We show that inclusion of LNA bases in antisense ONs augments the interference of HIV-1 genome dimerization. We also demonstrate the concomitant RNase H activation by six consecutive DNA bases in an LNA/DNA mix-mer. We show ON uptake via receptor-mediated transfection of a human T-cell line in which the mix-mers subsequently inhibit replication of a clinical HIV-1 isolate. Thus, the technique of LNA/DNA mix-mer antisense ONs targeting the conserved HIV-1 DIS region may provide a strategy to prevent HIV-1 assembly in the clinic.     

Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication

West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH₂ inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11?µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.     

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.     

Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.     

猪β干扰素在毕赤酵母中的分泌表达及其对伪狂犬病毒的抑制作用

为了研制高活性的重组猪β干扰素,对PoIFN-β成熟蛋白第3、7和164位的3个氨基酸密码子进行毕赤酵母偏嗜性改造并构建了酵母表达载体pPICZαA-PIB。pPICZαA-PIB经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。多株PCR鉴定为阳性的酵母转化子经甲醇诱导发酵分泌表达了PoIFN-β,其中B1株酵母的PoIFN-β产量最高,约为2.5×105U/mL,其表达量约为60μg/mL,比活为4.17×106U/mg。将发酵上清液用PEG20000浓缩后进行SDS-PAGE和Western blot检测,结果表明表达产物是分子量约为28kDa和25kDa蛋白的混合物,两者均可与PoIFN-β阳性抗血清发生特异反应。表达产物比PoIFN-β理论推导分子量(约20.8kDa)大,推测可能是表达产物发生了不同程度的糖基化。重组PoIFN-β对伪狂犬病毒在细胞中增殖可呈现抑制作用,并且rPoIFN-β对伪狂犬病毒在MDBK细胞上早期增殖的抑制效果最为明显。     

Lipase of Candida albicans induces activation of NADPH oxidase and l-arginine pathways on resting and activated macrophages

Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (M?) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on M? functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting M?s and M?s primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.     

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.