Differential recruitment of B and T cells in coxsackievirus B4-induced pancreatitis is influenced by a capsid protein.

Two genetically similar variants of coxsackievirus B4, CB4-P and CB4-V, cause distinct disease syndromes in mice. A multidisciplinary approach was used to examine the events occurring in situ. The CB4-P variant induced acute pancreatitis, followed by repair of the exocrine tissues, while the CB4-V variant induced chronic pancreatitis, characterized by extensive destruction of the exocrine tissues. Since CB4-V replicated more efficiently than CB4-P in vivo, the more extensive tissue injury associated with CB4-V infection could be explained as the result of a higher level of viral replication. However, the fact that CB4-V replicated more efficiently in a mouse strain that survives infection than in a strain that succumbs to infection suggests that immune-mediated mechanisms as well as viral cytolysis may contribute to pancreatic tissue injury. To address the role of the immune system in virus-induced pancreatitis, the cell types within the inflammatory infiltrate were analyzed by flow cytometry. B cells (34 to 75%) were the most abundant, followed by T cells (10 to 30%), natural killer cells (4 to 8%), and macrophages (0 to 6%). Recruitment (and perhaps proliferation) of B and T cells to the pancreatic tissues was influenced by viral strain. Differential recruitment of T and B cells may reflect altered antigenic sites between CB4-P and CB4-V. The viral sequence that affected T- and B-cell recruitment was identified as a threonine residue at position 129 of the VP1 capsid protein.     

Progression or resolution of coxsackievirus B4-induced pancreatitis: a genomic analysis

Group B coxsackieviruses are associated with chronic inflammatory diseases of the pancreas, heart, and central nervous system. Chronic pancreatitis, which can develop from acute pancreatitis, is considered a premalignant disorder because it is a major risk factor for pancreatic cancer. To explore the genetic events underlying the progression of acute to chronic disease, a comparative analysis of global gene expression during coxsackievirus B4-induced acute and chronic pancreatitis was undertaken. A key feature of acute pancreatitis that resolved was tissue regeneration, which was accompanied by increased expression of genes involved in cell growth, inhibition of apoptosis, and embryogenesis and by increased division of acinar cells. Acute pancreatitis that progressed to chronic pancreatitis was characterized by lack of tissue repair, and the expression map highlighted genes involved in apoptosis, acinoductular metaplasia, remodeling of the extracellular matrix, and fibrosis. Furthermore, immune responses appeared skewed toward development of alternatively activated (M2) macrophages and T helper 2 (Th2) cells during disease that resolved and toward classically activated (M1) macrophages and Th1 cells during disease that progressed. Our hypothesis is that growth and differentiation signals coupled with the M2/Th2 milieu favor acinar cell proliferation, while diminished growth signals and the M1/Th1 milieu favor apoptosis of acinar cells and remodeling/proliferation of the extracellular matrix, resulting in fibrosis.     

Hormonal regulation of CD4(+) T-cell responses in coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infection causes significant cardiac inflammation in male, but not female, B1.Tg.Ealpha mice. This gender difference in disease susceptibility correlates with selective induction of CD4(+) Th1 (gamma interferon-positive) cell responses in animals with testosterone, whereas estradiol promotes preferential CD4(+) Th2 (interleukin-4 positive [IL-4(+)]) cell responses. Differences in immune deviation of CD4(+) T cells cannot be explained by variation in B7-1 or B7-2 expression. Infection significantly upregulated both molecules, but no differences were detected between estradiol- and testosterone-treated groups. Significantly increased numbers of activated (CD69(+)) T cells expressing the gammadelta T-cell receptor were found in male and testosterone-treated male and female mice. In vivo depletion of gammadelta+ cells by using monoclonal antibodies inhibited myocarditis and resulted in a shift from a Th1 to Th2 response phenotype. Taken together, our results indicate that testosterone promotes a CD4(+) Th1 cell response and myocarditis by promoting increased gammadelta+ cell activation.     

Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.     

Antiviral and myocyte protective effects of murine interferon-beta and -{alpha}2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice

The present study tested the hypothesis that murine (m)IFN-beta or mIFN-alpha(2) can eliminate cardiac viral load and protect cardiomyocytes from injury in animals infected with coxsackievirus B3 (CVB3). CVB3-inoculated male Balb/c mice exhibited signs of illness, including lethargy, progressive weight loss, and death (10% on day 3 and 100% on day 8). Cardiac viral load was high [4,277 +/- 1,009 plaque-forming units and 25 +/- 5 copies CVB3/hypoxanthine guanine phosphoribosyl transferase 1 mRNA] on day 4. The cardiac tissue exhibited severe inflammatory infiltration and myocyte damage with an average myocarditis integrated pathology score of 2.1 +/- 0.2 on day 7. Most of the mice infected with CVB3 also developed epicarditis, and 55% had intraventricular thrombi present. Treatment with mIFN-beta [2.5 to 10 million international units (MIU)/kg] dose-dependently improved the general health status in CVB3-inoculated mice, as evidenced by reduction in weight loss, prevention of death, elimination of cardiac viral load, protection of myocytes from injury, decrease in inflammatory cell infiltration, and attenuation of intraventricular thrombus formation. Treatment with 10 MIU/kg mIFN-alpha(2) resulted in a similar level of efficacy as that induced by 5 MIU/kg mIFN-beta, with the exception that mIFN-alpha(2) did not reduce cardiac CVB3 mRNA. However, mIFN-alpha(2) , but not any dose group of mIFN-beta, significantly attenuated CVB3-induced epicarditis. These data demonstrate antiviral effects for both mIFN-beta and mIFN-alpha(2), which lead to protection of the mice from CVB3-induced myocarditis. However, the potential mechanisms leading to a differential host response for the two isoforms of mIFN remain to be elucidated.     

Attenuating mutations in coxsackievirus B3 map to a conformational epitope that comprises the puff region of VP2 and the knob of VP3

Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5' nontranslated region (5'NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100- to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5'NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5'NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.     

Identification of a single amino acid residue in the capsid protein VP1 of coxsackievirus B4 that determines the virulent phenotype.

To identify the molecular determinants of virulence for coxsackievirus B4, a panel of recombinant, chimeric viruses were constructed from cDNA clones of a virulent virus, CB4-V, and a nonvirulent virus, CB4-P. Initial studies mapped a major determinant of virulence to the 5' end of the viral genome, which contained the 5' untranslated and the P1 regions (A. Ramsingh, A. Hixson, B. Duceman, and J. Slack, J. Virol. 64:3078-3081, 1990). To determine whether the 5' untranslated region contributed to the virulent phenotype, a chimeric virus (vCB403) containing this region of the virulent virus on an avirulent background was tested in mice. The vCB403 construct displayed a phenotype similar to that of CB4-P, suggesting that the 5' untranslated region did not significantly contribute to virulence. Analysis of the sequence data of the P1 regions of both CB4-V and CB4-P revealed five mutations that resulted in amino acid substitutions in VP1, VP2, and VP4 (A. Ramsingh, H. Araki, S. Bryant, and A. Hixson, Virus Res. 23:281-292, 1992). Analysis of individual mutations in both VP1 and VP2 revealed that a single residue (Thr-129 of VP1) determined the virulent phenotype.     

Treatment with natural human interferon alpha of a CML-patient with antibodies to recombinant interferon alpha-2b

A patient with Philadelphia-chromosome positive chronic myelogenous leukemia developed interferon antibodies on treatment with recombinant interferon alpha-2b. Clinically this event corresponded with progressive disease. No cross-reactivity of antibodies with human leukocyte interferon was found by Western blot. Treatment was switched to human leukocyte interferon with an obvious clinical effect: WBC was reduced and platelet count stabilized, but the effect was transient and no hematologic remission was achieved. Human leukocyte interferon may be an alternative in CML-patients with neutralizing antibodies to recombinant interferon alpha.     

Inhibition of alpha/beta interferon signaling by the NS4B protein of flaviviruses

Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-alpha/beta) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-alpha/beta. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.     

RIPK1- and RIPK3-induced cell death mode is determined by target availability

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a ‘switch'' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.Mammalian cells can use a number of mechanisms to kill themselves. The best characterized depends on the Bcl-2 family members Bax and Bak that work via mitochondria to activate caspases.¹ Some caspases, notably caspase 8, can be activated independently of Bcl-2 family members, for example, after stimulation of members of the TNF receptor superfamily.² Recently, it has become apparent that some of these receptors, including TNFR1, can activate a third suicide mechanism that does not require caspases, and in which the morphology of the dying cell differs from classical apoptosis. This form of cell death, termed ‘necroptosis'', can often be blocked by necrostatin-1 (nec-1), an inhibitor of the kinase activity of receptor-interacting protein kinase 1 (RIPK1).^{3, 4} Accordingly, observations from several groups have shown that in some cell types, expression of RIPK1 can signal cell death by caspase-independent necroptosis.⁵It has previously been revealed that RIPK1 could function downstream of death receptors, but in those cases, cell death was usually blocked by coexpression of the viral inhibitor of caspases 1 and 8, CrmA,⁶ and typically exhibited a classical ‘apoptotic'' morphology. It was revealed that RIPK1 engages FADD via homotypic binding of their death domains (DDs), and FADD in turn activates caspase 8.^{6, 7}RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD.^{8, 9, 10, 11} RIPK3 is required for necroptosis.^{12, 13} Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1.¹⁴ RIPK3 activates, by phosphorylation, MLKL, a pseudokinase essential for this death pathway.^{15, 16, 17} Once activated, MLKL forms multimers that trigger breaches of the plasma membrane.^{18, 19, 20}Although RIPK3 is necessary for necroptosis, it is unclear whether activation of RIPK3 is sufficient for cell death, because TNF activates signaling by many pathways in addition to those controlled by RIPK1.²¹ It is also unclear whether RIPK3 can contribute to apoptosis. Despite some reports to this effect,^{8, 9, 22} RIPK3 has been described as the necroptotic ‘switch'', implying its activity precipitates necroptosis to the exclusion of apoptosis.^{23, 24, 25}Here, we have directly activated RIP kinases without the confounding effects of multiple signals emanating from the target cell''s cytokine receptors, allowing us to define more precisely the functions of RIPK1 and RIPK3. We activated RIP kinases by dimerization using inducible lentiviral vectors, each encoding a chimera of a RIP kinase with subunit B of E. coli DNA gyrase.²⁶ We infected mouse embryonic fibroblasts (MEFs) that lack genes for, or expression of, various cell death proteins, induced expression of the RIPK chimera, caused its dimerization by addition of the divalent antibiotic coumermycin (C) and quantitated the resulting cell death.Our results reveal that each of RIPK1 and RIPK3 can contribute to both apoptosis and necroptosis depending on the biochemical context. Furthermore, necroptosis can occur in the absence of caspase 8 and FADD, which shows that the ripoptosome, with core components caspase 8, FADD and RIPK1,^{27, 28} is not required for necroptosis. Instead, we propose that dimers of RIPK1 and/or RIPK3 are the pivotal complexes from which both forms of cell death can progress.     

Diabetes acceleration or prevention by a coxsackievirus B4 infection: critical requirements for both interleukin-4 and gamma interferon

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-gamma) genes (NOD IL-4-/- and NOD IFN-gamma-/-, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-gamma delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-gamma-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-gamma-/- mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4-/- mice, while neither acceleration nor long-term protection was elicited in NOD IFN-gamma-/- mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4-/-, and NOD IFN-gamma-/- mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-gamma contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-gamma.     

Viral entry through CXCR4 is a pathogenic factor and therapeutic target in human immunodeficiency virus type 1 disease

The chemokine receptors CCR5 and CXCR4 function as the principal coreceptors for human immunodeficiency virus type 1 (HIV-1). Coreceptor function has also been demonstrated for a variety of related receptors in vitro. The relative contributions of CCR5, CXCR4, and other putative coreceptors to HIV-1 disease in vivo have yet to be defined. In this study, we used sequential primary isolates and recombinant strains of HIV-1 to demonstrate that CXCR4-using (X4) viruses emerging in association with disease progression are highly pathogenic in ex vivo lymphoid tissues compared to CXCR4-independent viruses. Furthermore, synthetic receptor antagonists that specifically block CXCR4-mediated entry dramatically suppressed the depletion of CD4(+) T cells by recombinant and clinically derived X4 HIV-1 isolates. Moreover, in vitro specificity for the additional coreceptors CCR3, CCR8, BOB, and Bonzo did not augment cytopathicity or diminish sensitivity toward CXCR4 antagonists in lymphoid tissues. These data provide strong evidence to support the concept that adaptation to CXCR4 specificity in vivo accelerates HIV-1 disease progression. Thus, therapeutic intervention targeting the interaction of HIV-1 gp120 with CXCR4 may be highly valuable for suppressing the pathogenic effects of late-stage viruses.     

Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from envenomation. We suggest that the A1BG-CRISP-3 complex displays a similar function in protecting the circulation from a potentially harmful effect of free CRISP-3.     

Design,synthesis, and biological evaluation of novel coxsackievirus B3 inhibitors

The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These compounds could be considered as the 6-chloropurines substituted at position 9 with variously substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene—norbornane or norbornene). The synthesis and biological evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in the low micromolar range (0.66–2 μM). Minimal or no cytotoxicity was observed.     

Inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle

Despite replicating to very high titers, coxsackieviruses do not elicit strong CD8 T-cell responses, perhaps because antigen presentation is inhibited by virus-induced disruption of host protein trafficking. Herein, we evaluated the effects of three viral nonstructural proteins (2B, 2BC, and 3A) on intracellular trafficking. All three of these proteins inhibited secretion, to various degrees, and directly associated with the Golgi complex, causing trafficking proteins to accumulate in this compartment. The 3A protein almost completely ablated trafficking and secretion, by moving rapidly to the Golgi, and causing its disruption. Using an alanine-scanning 3A mutant, we show that Golgi targeting and disruption can be uncoupled. Thus, coxsackieviruses rely on the combined effects of several gene products that target a single cellular organelle to successfully block protein secretion during an infection. These findings have implications for viral pathogenesis.     

Purification and properties of a novel recombinant human hybrid interferon, delta-4 alpha 2/alpha 1

The human interferon (huIFN) delta-4 alpha 2(5-62)/alpha 1(64-166) is a genetically engineered hybrid that consists of residues 5-62 of huIFN alpha 2 and residues 64-166 of huIFN alpha 1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN alpha subtypes. This novel recombinant hybrid was purified from Escherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17,800 and 17,100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7 x 10(7) units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) on various cell lines of different histological origin appeared to be more comparable to that of huIFN alpha 1 than huIFN alpha 2. The data suggest that huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) hybrid may be a useful tool for understanding huIFN structure-function relations.