An antiviral substance from Penicillium funiculosum. V. Induction of interferon by helenine.

Helenine, a substance obtained from broth cultures of Penicillium funiculosum and known to exert a protective effect in vivo in experimental animals against several unrelated viruses, has been shown to elicit the formation in cell cultures and in intact mice of an inhibitor of viral plaque formation. Because the biological and chemical characteristics of the viral inhibitor induced by helenine are similar to those of interferon, it is suggested that the antiviral effect of helenine may be mediated through the formation of interferon.

Maturation, activation, and protection of dendritic cells induced by double-stranded RNA.

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.

An antiviral soluble form of the LDL receptor induced by interferon.

Interferons, which induce several intracellular antiviral proteins, also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This 28-kilodalton soluble protein was purified to homogeneity and identified by protein sequencing as the ligand-binding domain of the human 160-kilodalton low density lipoprotein receptor (LDLR). The existence of an antiviral soluble LDLR was confirmed by immunoaffinity chromatography with monoclonal antibody to LDLR. This soluble receptor mediates most of the interferon-triggered antiviral activity against VSV, apparently by interfering with virus assembly or budding, and not by inhibiting virus attachment to cells.

Use of an indirect pharmacodynamic stimulation model of MX protein induction to compare in vivo activity of interferon alfa-2a and a polyethylene glycol-modified derivative in healthy subjects.

Interferon alfa-2a was chemically modified by the covalent attachment of a polyethylene glycol (PEG) moiety to enhance its circulating half-life and to reduce its immunogenicity. A comparative evaluation of the pharmacokinetics of the PEG-modified interferon alfa-2a showed a greater than twofold increase in the circulating half-life as a result of this chemical modification. An indirect physiologic response model was developed to characterize the time course of the MX protein response after subcutaneous administration of single ascending doses of either interferon alfa-2a or PEG-interferon alfa-2a in healthy volunteers. Analysis of the pharmacokinetic-pharmacodynamic relationship suggested that the PEG-modified interferon alfa-2a could not be administered less than twice weekly and therefore offered little therapeutic advantage over its unmodified counterpart, which is administered three times weekly. These results were consistent with findings in phase II trials. This study substantiates the usefulness of pharmacodynamic modeling as a tool for the development of dose recommendations and for the early selection of drug candidates in the drug development process.

The interferon-induced Mx protein of chickens lacks antiviral activity.

cDNA sequencing revealed that chick Mx protein consists of 705 amino acids. Its 84 N-terminal amino acids show no significant sequence homology to other Mx proteins. They are followed by 514 residues that include a tripartite GTP binding consensus motif. This region shows 50-70% sequence identity to mammalian and duck Mx proteins. Sequences near the C terminus, including a leucine zipper motif, are also conserved, whereas the intervening 19 amino acids lack sequence similarity. This unique sequence corresponds to a highly variable region in mammalian Mx proteins, suggesting that it serves as a spacer between functional domains. Chick and mouse cells transiently transfected with cDNA expression constructs synthesized chick Mx protein at a level that could easily be detected with specific antibodies. Chick Mx protein in such cells was mainly cytoplasmic and had a granular appearance. Permanently transfected cell lines expressing high levels of chick Mx protein could not be established, suggesting low metabolic stability of chick Mx protein or incompatibility with cell proliferation. The antiviral activity of chick Mx protein was tested at the single-cell level using immunofluorescence techniques. Transfected cells expressing chick Mx protein showed no enhanced resistance to influenza A virus, vesicular stomatitis virus, Thogoto virus, or Sendai virus. Thus, chick Mx joins the list of Mx proteins without recognized antiviral activity, supporting the concept that Mx proteins serve unrelated functions.

A comparative investigation of CC chemokines and SIV suppressor factors generated by CD8+ and CD4+ T cells and CD14+ monocytes.

The capacity of CD8+ and CD4+ T cells and CD14+ monocytes to generate the CC chemokines, RANTES, MIP-1alpha and MIP-1beta, and SIV suppressor factors were studied using cells separated from PBMC of macaques immunized with the 70-kDa heat shock protein (HSP70). Unimmunized macaques showed low levels of the three CC chemokines and SIV-SF, and they showed little variation between PBMC and the two subsets of T cells stimulated with PHA. Immunization with HSP70 elicited an increase in the in vitro concentration of each of the three CC chemokines and SF. This was found with PBMC, CD4+ and CD8+ T cells and to a lesser extent with monocytes, when conventionally separated enriched cell subsets were examined from the same PBMC. However, the concentrations of the three CC chemokines derived from highly purified cell-sorted populations (>95%) were greatly increased, as compared with the enriched cell subsets. The concentration of each of the three chemokines was highest for CD8+ T cells, decreased with CD4+ T cells and was lowest with the CD14+ monocytes, but the latter were not stimulated. Neutralization assays with antibodies to the three CC chemokines showed that the antiviral activity generated by the four populations of cells could be largely accounted for by the three CC chemokines. The results of this comparative study suggests that CD8+ as well as CD4+ T cells and CD14+ monocytes generate the three CC chemokines and SIV-SF when stimulated with a mitogen, and that the baseline innate level can be upregulated by adaptive immune responses to a specific antigen.

Molecular cloning of double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.).

Mx proteins are induced by type I interferons in mice and humans and inhibit the replication of several negative-stranded RNA viruses. In this work Mx genes in Atlantic salmon were studied using the double stranded RNA, polyinosinic: polycytidylic acid (poly I:C), to induce interferon production. Northern blot analysis showed Mx mRNA in liver, head kidney and gills 2 days after poly I:C injection of fish, but not in untreated fish or fish injected with saline or LPS. Mx transcripts of 2.4 and 1.9 kb were detected in the liver. By screening of a cDNA library, three different full length Mx cDNA clones, ASMx1, ASMx2 and ASMx3, were identified and sequenced. The deduced ASMx proteins all contain 623 amino acids and show the tripartite GTP-binding motif typical of vertebrate Mx proteins. ASMx1 and ASMx2 may represent alleles of the same gene whereas ASMx3 represents a different gene. The deduced ASMx proteins showed 96 to 98% sequence identity with rainbow trout Mx1 and Mx3 and about 88% identity with rainbow trout Mx2 protein.

Proteins induced by recombinant equine interferon-beta 1 within equine peripheral blood mononuclear cells and polymorphonuclear neutrophilic granulocytes.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in immunoblot tests. The 74 kDa and perhaps the 82 kDa components may thus represent equine 'Mxanalogous proteins'. The 74 kDa protein was only detected in PBMC of ten out of 20 horses examined. The induction of Mx protein in the horse by Type 1 interferon may therefore resemble that in the mouse, where Mx protein is involved in selective resistance to influenza virus. The influence of rEqIFN-beta 1 on protein expression in equine PBMC and PMN was monitored by metabolic labeling and 2-D gel electrophoresis. Proteins of 82, 74, 58 and 40 kDa were induced in PBMC following exposure to rEqIFN-beta 1. A constitutively expressed 35 kDa protein, however, was no longer demonstrable upon treatment with interferon. None of the proteins induced within PBMC was found in highly purified PMN treated with interferon. PMN exposed to rEqIFN-beta 1 synthesized four proteins in the range of 25 to 27 kDa. These proteins have not been described in interferon-treated PMN of any other species.

Endogenous salivary inhibitors of human immunodeficiency virus.

The human immunodeficiency virus type 1 (HIV-1) is rarely transmitted through salivary secretions, due in part to the presence of endogenous inhibitors. Here, the protective characteristics of the intraoral environment are summarized and inhibitory factors that reduce HIV-1 infectivity in vitro described, focusing on secretory leucocyte protease inhibitor (SLPI), a 12-kDa mucosal protein that blocks HIV infection in several cell-culture systems. SLPI appears to interact with a cellular surface molecule to limit viral entry into target cells. To determine whether the inhibitor has a similar role in vivo, the contribution of salivary SLPI to anti-HIV-1 activity was assessed. Whole unstimulated filtered salivas from infected and uninfected donors contained similar concentrations of the inhibitor. Depletion from SLPI filtered saliva produced a corresponding loss of inhibitory activity. In general, filtered whole salivas obtained from 10 donors had antiviral activities that correlated positively with SLPI concentrations. However, some samples having SLPI well below the concentration required for inhibitory activity in vitro exhibited modest inhibition, suggesting the presence of other anti-HIV-1 components in oral fluids. Thus, SLPI is a major but not sole inhibitor of this virus in saliva.

Lanthiopeptin, a new peptide antibiotic. Production, isolation and properties of lanthiopeptin.

A strain of Streptoverticillium cinnamoneum produced a peptide antibiotic named lanthiopeptin, which contained four unusual amino acids, erythro-beta-hydroxyaspartic acid, mesolanthionine, threo-beta-methyllanthionine and lysinoalanine. Lanthiopeptin showed antiviral activity against herpes simplex virus type 1 KOS strain infection in Vero cells by cytopathic effect reduction assay. The structure of lanthiopeptin is similar to that of ancovenin.

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV.

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.

Comparative analysis of interferon and antiviral protein messenger RNAs.

A comparative analysis of interferon and antiviral protein messenger RNAs was carried out. Differences in their biological activities and sedimentation coefficients were found. In RNA preparations from superinduced cells (cells treated with poly(I).poly(C) and antimetabolites) and from cells treated with interferon, messenger RNAs possesing interferon and antiviral activities were detected. The results suggest the existence of two types of mRNA (for interferon and antiviral protein, respectively) and support the hypothetic model of interferon action via an antiviral protein.

[Alpha interferon, antiviral proteins and their value in clinical medicine]. / Interféron alpha, protéines antivirales et applications médicales.

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.

[Proof of the necessity of antiviral protein synthesis in the presence of interferon action]. / Dokazatel'stvo neobkhodimosti sinteza antivirusnogo belka pri deistvii interferona

The previously described cell-free system of combined synthesis of virus components was used for investigation of the mechanism of interferon action. Interferon effect was demonstrated to require the cellular stage of its action. When interferon was added after separation of the microsomal-mitochondrial "factory", no inhibitory effect was observed. The experimental data confirm the concept that interferon induces an antiviral protein coded in cell genome.

[Identification of the isoadenylate synthetase in the messenger RNA translation products of antiviral proteins]. / Identifikatsiia izoadenilatsintetazy v produktakh transliatsii informatsionnykh RNK antivirusnykh belkov.

A further study of conditions for induction, determinations of activity and products of mRNAs translation of AVP having nonspecific antiviral activity from L-929 cells treated with poly(I) . poly(C) was carried out. Under conditions of superinduction of interferon the yield of AVP mRNA is reduced unlike that of interferon mRNA. Manifestations of the antiviral effect of AVP mRNA translation products do not seem to require the stages of transcription as dihydrorifampicin exerts no influence on this process. The inhibiting effect of actinomycin D is more likely to be associated with its effect on the energy of cells and synthesis of macroergic compounds of ATP type. Active AVP mRNAs from L-929 cells like interferon mRNA consist mainly of poly-A-deficient mRNAs. The sedimentation rate of AVP mRNAs differs from that of interferon mRNA and is about 10S. AVP mRNA translation products in ovocytes of Xenopus laevis, in contrast to control mRNAs, contain isoadenylate synthetase responsible for synthesis of 2'5'-oligoisoadenylate, a nonspecific translation inhibitor the mechanism of action of which consists in activation of cellular endogenous nuclease. The results suggest that the mode of development of the antiviral condition by activation of isoadenylate synthetase is similar in the cells treated with interferon and poly(I) . poly(C).