Viral protein VP4 is a target of human antibodies enhancing coxsackievirus B4- and B3-induced synthesis of alpha interferon

Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-alpha) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-alpha were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56 degrees C from CVB4E2 (VP4(CVB4)) and CVB3 (VP4(CVB3)) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis. There was no cross-reaction between VP4(CVB4) and VP4(CVB3) in the inhibiting effect. IFN-alpha levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4(CVB4)). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-alpha levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis by PBMC.     

Interferon induces tryptophanyl-tRNA synthetase expression in human fibroblasts.

A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.     

Neutralizing Activity Induced by the Attenuated Coxsackievirus B3 Sabin3-like Strain Against CVB3 Infection

Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. In the present work, we assessed whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and CVB4 E2 strains in mice by both oral and intraperitoneal (IP) routes. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their anti-CVB3 neutralizing activity. CVB3 Sabin3-like was highly attenuated in vivo and was able to induce an anti-CVB3 activity of the serum. However, at 4 days post-CVB3 challenge, significant increased titers of CVB3 neutralizing antibodies were detectable in the sera of immunized mice over the next 6 days. Non-immunized mice challenged with CVB3 Nancy had no anti-CVB3 activity in their sera until 10 days post-infection. CVB3 Nancy induced higher viral titers than did the mutant strain. There was no variation of the neutralizing activity of serum taken from mice immunized with CVB3 Sabin3-like and challenged with CVB4 E2, compared to non-immunized mice. Despite the fact that CVB3 and CVB4 are closely related viruses, virus-neutralizing activity clearly distinguish between these viruses. A variable and limited amount of pancreatic inflammation was seen in some mice 10 days after Sabin3-like inoculation by IP route, whereas there was no evidence of pancreatic damage in mice inoculated by oral route. All immunized mice were protected from myocarditis and pancreatitis at 8 days post-challenge with CVB3 or CVB4 E2. These findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.     

Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.     

IFN-zeta/ limitin: a member of type I IFN with mild lympho-myelosuppression

Interferon (IFN)-Zeta/limitin has been considered as a novel type I IFN by the Nomenclature Committee of the International Society for Interferon and Cytokine Research. IFN-Zeta/limitin shows some sequence homology with IFN-alpha and IFN-beta, has a globular structure with five alpha-helices and four loops, and recognizes IFN-alpha/beta receptor. Although IFN-zeta/limitin displays antiviral, immunomodulatory, and antitumor effects, it has much less lympho-myelosuppressive activities than IFN-alpha. Treatment of cells with type I IFNs induces and/or activates a number of molecules, which regulate cell cycle and apoptosis. It is noteworthy that IFN-zeta/limitin activates the Tyk2-Daxx and Tyk2-Crk pathways weaker than IFN-alpha. Because experiments using antisense oligonucleotides have revealed their essential role in type I IFN-related suppression of lympho-hematopoiesis, little ability of IFN-zeta/limitin to activate the Tyk2-dependent signaling pathway may explain its uniquely narrow range of biological activities. Further analysis of structure-function relationship of type I IFNs will establish an engineered cytokine with useful features of IFN-zeta/limitin.     

RNase L and double-stranded RNA-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection

Coxsackievirus (CV) is an important human pathogen that has been linked to the development of autoimmunity. An intact pancreatic beta cell IFN response is critical for islet cell survival and protection from type 1 diabetes following CV infection. In this study, we show that IFNs trigger an antiviral state in beta cells by inducing the expression of proteins involved in intracellular antiviral defense. Specifically, we demonstrate that 2',5'-oligoadenylate synthetases (2-5AS), RNase L, and dsRNA-dependent protein kinase (PKR) are expressed by pancreatic islet cells and that IFNs (IFN-alpha and IFN-gamma) increase the expression of 2-5AS and PKR, but not RNase L. Moreover, our in vitro studies uncovered that these pathways play important roles in providing unique and complementary antiviral activities that critically regulate the outcome of CV infection. The 2-5AS/RNase L pathway was critical for IFN-alpha-mediated islet cell resistance from CV serotype B4 (CVB4) infection and replication, whereas an intact PKR pathway was required for efficient IFN-gamma-mediated repression of CVB4 infection and replication. Finally, we show that the 2-5AS/RNase L and the PKR pathways play important roles for host survival during a challenge with CVB4. In conclusion, this study has dissected the pathways used by distinct antiviral signals and linked their expression to defense against CVB4.     

A group B coxsackievirus/poliovirus 5' nontranslated region chimera can act as an attenuated vaccine strain in mice

The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5' NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.     

Bacillus alcalophilus peptidoglycan induces IFN-alpha-mediated inhibition of vaccinia virus replication

Bacterial products such as cell walls (CW) and peptidoglycan (PGN) are known to activate macrophages and NK cells during microbial infections. In this report, we demonstrated that whole CW and PGN of four Gram-positive bacteria are capable of enhancing the anti-poxviral activity of murine macrophage RAW 264.7 cells. Among the major Bacillus alcalophilus CW components, PGN contributes the most to antiviral activity and induces remarkably higher levels of IFN-alpha. Anti-IFN-alpha/beta antibody, but not anti-IFN-gamma, anti-IFN-gamma receptor, or anti-IL-12, reversed the PGN-induced inhibition of vaccinia virus replication and reduced nitric oxide (NO) production. Our data thus suggest that PGN induce antiviral activity through IFN-alpha and to a lesser extent, through NO production.     

Penetration and Antiviral Activity of Coxsackievirus B3 (Cvb3)-Specific Phosphorothioate Oligodeoxynucleotides (Ps-Odn)

Abstract

The antiviral activity of PS-ODNs, complementary to different regions of the CVB3 genome, was investigated under in vitro conditions. Inhibition of CVB3 replication was detected only after prolonged pretreatment of HeLa cells with antiviral active PS-ODNs, but not when virus and PS-ODN were applicated simultaneously. Results from flow cytometric analysis indicate that a low cellular uptake of anti-CVB3 oligonucleotides into HeLa cells might be a reason for their moderate antiviral activity.     

A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge

We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.     

Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-alpha, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.     

Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon.

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.     

Induction of an Antiviral State and Attenuated Coxsackievirus Replication in Type III Interferon-Treated Primary Human Pancreatic Islets

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets'' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.     

A large component of the antiviral activity of mouse interferon-gamma may be due to its induction of interferon-alpha

The finding that interferon-gamma (IFN-gamma) may require two rounds of protein synthesis to induce the antiviral state raises the possibility that this IFN may not be directly antiviral. We, therefore, examined the possibility that IFN-gamma induces one or both of the other IFNs (alpha and/or beta) which in turn induce the antiviral state. Evidence is presented showing that under certain conditions a large portion of IFN-gamma's antiviral activity in mouse L-929 cells is mediated by its induction of IFN-alpha based on the findings that: 1) the antiviral activity of IFN-gamma in cells at low densities can be blocked by poly and monoclonal antibody to IFN-alpha and, 2) IFN-alpha can be demonstrated in the supernatant fluids of IFN-gamma treated cells. This report raises the possibility that a major antiviral mechanism of IFN-gamma is via induction of IFN-alpha in the mouse system. If the majority of the antiviral activity of IFN-gamma is via induction of other IFNs, then the role and mechanism of IFN-gamma might have to be reevaluated.