Philip I. Marcus

Interferon Induction by Viruses

Interferon was discovered when Isaacs and Lindenmann (1957) infected fragments of chick chorioallantoic membrane with heat-inactivated influenza virus. The viral component or components responsible for interferon induction were not identified then, and indeed the inducer remains unknown a quarter-century later. Isaacs et al. (1963) postulated that the molecular species responsible for interferon induction was “foreign nucleic acid”. In 1967 that “foreign nucleic acid” took the form of double-stranded RNA (dsRNA) when Field et al. (1967) discovered that exogenously added synthetic polyribonucleotides were inducers of interferon. There followed a plethora of studies designed to relegate to viral dsRNA the comparable role of interferon inducer in the virus-infected cell. Many of the studies provided evidence in support of viral dsRNA as the interferon inducer moiety, yet many others suggested that dsRNA itself did not induce interferon and that some additional early viral replicative events were required. In the extreme, interferon induction was reported to occur in cells infected with inactivated viruses and under conditions where dsRNA was not detectable. In addition, the apparent lack of correlation between the replication of viral RNA and interferon induction in several systems again pointed to the possibility that the accumulation of viral dsRNA might not suffice to induce interferon. To complicate matters, some investigators reported inverse correlations between viral RNA replication and interferon induction. Even virions containing preformed dsRNA did not always function as inducers of interferon. Confronted with these apparently conflicting data, it was reasonable to conclude that either dsRNA was not the molecule responsible for interferon induction, or that additional factors were involved. Johnston and Burke (1973) and Stewart (1979) very succinctly review and astutely interpret these earlier studies. Against this background of conflicting reports we initiated a series of experiments designed to define the molecule or molecules of viral origin responsible for interferon induction (Marcus and Sekellick 1977). We report these and related results herein.

Interferon Action: Inhibition of Vesicular Stomatitis Virus RNA Synthesis Induced by Virion-Bound Polymerase

The particle-bound RNA polymerase activity of vesicular stomatitis virus (VSV) can be demonstrated in vivo. Linear synthesis of viral RNA persists for 5 to 6 hours at 34�C in infected monolayers of chick embryo cells treated with cycloheximide and actinomycin D to block synthesis of protein and cell-specific RNA. At least 55 percent of the RNA made under these conditions is complementary to virion RNA. RNA synthesis mediated by VSV polymerase activity is inhibited in cells first treated with chick-derived interferon or polyriboinosinate• polyribocytidylate, but not by mouse interferon. The RNA product of VSV polymerase activity is present throughout the cytoplasm, and its synthesis is inhibited by the interferon system, as judged by autoradiographs that show the physical distribution, in cells, of RNA produced by virion polymerase in the absence of translation—a demonstration of the transcription product of the viral genome.

[18] Induction of high titer chicken interferon

Publisher Summary This chapter provides a guide to produce high titers of chicken interferon (IFN). Primary cultures of chicken (chick) embryo cells can be used to produce high titers of chick interferons. However, to obtain high titers routinely, care must be exercised during the initial preparation of the primary cells from the embryos, incubation and treatment of the cultures, and the induction process per se. When all three of these conditions are optimized, a properly prepared, “aged,” and induced culture of primary chick embryo cells (CEC) can produce tens of thousands of units of IFN. Primary chick embryo cell (CEC) cultures are prepared from 10-day-old embryonated chicken eggs. Embryonated eggs from other sources and with different genetic backgrounds (e.g., scaleless mutant) have all produced primary CEC cultures capable of producing high titers of chicken IFN. Ten-day-old embryonated eggs are used, because, in general, younger embryos yield significantly fewer cells; those that are much younger than nine days old produce less IFN. Embryos older than 10 days produce primary cell cultures that yield high titers of IFN, but embryos older than 13 days are more difficult to mince and process for cell plating. To achieve and maintain multiplicities for optimal induction of IFN when using an infectious virus in a permissive cell, it is necessary to restrict the spread of infectious virus.

Cell killing by viruses. I. Comparison of cell-killing, plaque-forming, and defective-interfering particles of vesicular stomatitis virus.

Abstract Single-cell survival curves generated by infecting the Vero line of green monkey kidney cells, or mouse L cells with vesicular stomatitis virus (VSV) manifest oneparticle-to-kill kinetics at low multiplicities, permitting accurate determination of the cell-killing particle activity of a virus preparation. Cells adsorbing lethal amounts of VSV attach and spread normally on to growth surfaces. Cell killing assays demonstrate that VSV stocks may contain 5 times more cell-killing particles than plaqueforming particles. Noninfectious particles that kill cells are considered defective cell killing particles. Purification of standard B particles by successive velocity sedimentations in sucrose gradients produced relatively little loss of plaque forming or cell killing titer. Defective particles processed similarly showed a progressive decrease in plaque-forming and cell-killing activity, while maintaining close to the original titers of defective-interfering particles. Experiments with thrice gradient purified cell-killing particles and defective-interfering particles demonstrate that defective-interfering particles do not kill cells, nor do they interfere with the action of cell-killing particles, although they delay slightly the expression of cytopathicity. Cell killing by crude preparations of defective-interfering particles is attributable to contaminating cell-killing particles. The existence of a significant fraction of cellkilling particles that is noninfectious, and the lack of cell killing by purified, but active, defective-interfering particles, presumably containing all virion proteins, provide evidence that cell killing by VSV is not dependent upon complete viral replication, and that the virion per se is not inherently toxic. Proper virion-associated transcription is proposed as a requisite initial event in the molecular reactions leading to cell death.

Chicken Interferon Type I Inhibits Infectious Bronchitis Virus Replication and Associated Respiratory Illness

Infectious bronchitis virus (IBV) causes an economically important respiratory disease in poultry worldwide. Previous studies have shown that CD8(+) cytotoxic T lymphocytes (CTL) are critical in controlling acute IBV infection, but the role of innate immunity is unknown. This study describes the in vitro and in vivo anti-IBV activity of natural spleen cell-derived and recombinant chicken interferon type I (rChIFN-alpha). Both natural and rChIFN-alpha inhibited replication of the Beaudette strain of IBV in chicken kidney cells (CKC) in a dose-dependent manner, with the antiviral activity of the former accounted for entirely by its content of type I IFN. IFN at 100 U/ml reduced viral replication by 50% as measured by syncytia formation. In addition, the spleen cell-derived supernatants (natural IFN) inhibited tracheal ring ciliostasis mediated by the Gray strain of IBV. Optimal protection against IBV-induced respiratory disease was obtained after intravenous or oral administration of ChIFN given 1 day before virus challenge and each of 5 days thereafter. ChIFN-I protected chicks from clinical illness by delaying the onset of the disease and decreasing the severity of illness, demonstrating its potential as an immune enhancer.

Amelioration of Influenza Virus Pathogenesis in Chickens Attributed to the Enhanced Interferon-Inducing Capacity of a Virus with a Truncated NS1 Gene

ABSTRACT Avian influenza virus (AIV) A/turkey/Oregon/71-SEPRL (TK/OR/71-SEPRL) (H7N3) encodes a full-length NS1 protein and is a weak inducer of interferon (IFN). A variant, TK/OR/71-delNS1 (H7N3), produces a truncated NS1 protein and is a strong inducer of IFN. These otherwise genetically related variants differ 20-fold in their capacities to induce IFN in primary chicken embryo cells but are similar in their sensitivities to the action of IFN. Furthermore, the weak IFN-inducing strain actively suppresses IFN induction in cells that are otherwise programmed to produce it. These phenotypic differences are attributed to the enhanced IFN-inducing capacity that characterizes type A influenza virus strains that produce defective NS1 protein. The pathogenesis of these two variants was evaluated in 1-day-old and 4-week-old chickens. The cell tropisms of both viruses were similar. However, the lesions in chickens produced by the weak IFN inducer were more severe and differed somewhat in character from those observed for the strong IFN inducer. Differences in lesions included the nature of inflammation, the rate of resolution of the infection, and the extent of viral replication and/or virus dissemination. The amelioration of pathogenesis is attributed to the higher levels of IFN produced by the variant encoding the truncated NS1 protein and the antiviral state subsequently induced by that IFN. The high titer of virus observed in kidney tissue (≈109 50% embryo lethal doses/g) from 1-day-old chickens infected intravenously by the weak IFN-inducing strain is attributed to the capacity of chicken kidney cells to activate the hemagglutinin fusion peptide along with their unresponsiveness to inducers of IFN as measured in vitro. Thus, the IFN-inducing capacity of AIV appears to be a significant factor in regulating the pathogenesis, virulence, and viral transmission of AIV in chickens. This suggests that the IFN-inducing and IFN induction suppression phenotypes of AIV should be considered when characterizing strains of influenza virus.

Interferon induction by viruses. XVI. 2-Aminopurine blocks selectively and reversibly an early stage in interferon induction.

A purine analogue, 2-aminopurine, reported to act as an inhibitor of protein kinase, selectively, reversibly and in a dose-dependent manner blocked a very early stage in interferon induction. With chick embryo cells and mouse L cells as hosts, and different viral inducers of interferon, maximal effects of 2-aminopurine were observed during the first 4 h of induction. At 10 mM-2-aminopurine there was a 20-fold reduction in the yield of interferon from both cell types. 2-Aminopurine and actinomycin D both prevented interferon induction with the same time course, indicating a transcriptional block to induction; however, only the action of the former was reversed upon removal of the drug. Addition of 2-aminopurine to an agarose overlay resulted in high efficiency plaque formation by vesicular stomatitis virus New Jersey (Hazelhurst) under conditions where endogenous induction of interferon and its feedback action on aged chick embryo cells normally prevented plaque formation. Two other inducible systems, representing genes involved in interferon action (both its development and activation), and those of heat shock, were not affected by 2-aminopurine. A model is presented implicating the interferon-inducible dsRNA-dependent protein kinase as an interferon induction receptor which, on interaction with dsRNA, generates an amplified signal via phosphorylation that ultimately derepresses the interferon gene(s).

Interferon Induction and/or Production and Its Suppression by Influenza A Viruses

Developmentally aged chicken embryo cells which hyperproduce interferon (IFN) when induced were used to quantify IFN production and its suppression by eight strains of type A influenza viruses (AIV). Over 90% of the IFN-inducing or IFN induction-suppressing activity of AIV populations resided in noninfectious particles. The IFN-inducer moiety of AIV appears to preexist in, or be generated by, virions termed IFN-inducing particles (IFP) and was detectable under conditions in which a single molecule of double-stranded RNA introduced into a cell via endocytosis induced IFN, whereas single-stranded RNA did not. Some AIV strains suppressed IFN production, an activity that resided in a noninfectious virion termed an IFN induction-suppressing particle (ISP). The ISP phenotype was dominant over the IFP phenotype. Strains of AIV varied 100-fold in their capacity to induce IFN. AIV genetically compromised in NS1 expression induced about 20 times more IFN than NS1-competent parental strains. UV irradiation further enhanced the IFN-inducing capacity of AIV up to 100-fold, converting ISP into IFP and IFP into more efficient IFP. AIV is known to prevent IFN induction and/or production by expressing NS1 from a small UV target (gene NS). Evidence is presented for an additional downregulator of IFN production, identified as a large UV target postulated to consist of AIV polymerase genes PB1 + PB2 + PA, through the ensuing action of their cap-snatching endonuclease on pre-IFN-mRNA. The products of both the small and large UV targets act in concert to regulate IFN induction and/or production. Knowledge of the IFP/ISP phenotype may be useful in the development of attenuated AIV strains that maximally induce cytokines favorable to the immune response.

Hemadsorption-Negative Plaque Test: New Assay for Rubella Virus Revealing a Unique Interference tion-negative plaque technique

A simple and rapid plaque procedure has been developed for detecting and accurately assaying rubella virus in a noncytopathic virus-cell relationship. Plaque-formation is based on the development, in individual cells infected with rubella virus, of a unique type of intrinsic interference to infection with Newcastle disease virus. Rubella virus—infected cells challenged with Newcastle disease virus and tested for hemadsorption 15 hours later stand out as hemadsorption-negative areas. Individual living cells infected with rubella virus can be resolved under conditions allowing standard cloning procedures. In principle, the hemadsorption-negative plaque test can be used to search for a new class of noncytopathic, non-hemadsorbing viruses—those that induce an intrinsic interference to infection by any hemadsorbing virus.

Persistent infection II. Interferon-inducing temperature-sensitive mutants as mediators of cell sparing: Possible role in persistent infection by vesicular stomatitis virus

Abstract We demonstrate that like the interferon-inducing [±]RNA DI particles of vesicular stomatitis virus (VSV), ts mutants, also commonly implicated in the establishment and maintenance of persistent infection, can be excellent inducers of interferon—both at non-permissive (40°) and at semipermissive (37°) temperatures. Mutants capable of inducing interferon ( ifp + ) were characteristically negative or delayed in the expression of cellular protein synthesis inhibition— psi − and delayed psi + phenotypes, respectively. We contend that in cells competent for the interferon system, virus phenotypes selected during persistent infection are characterized by interferon-inducing particles ( ifp + ) activity. Interferon induction by ifp + mutants (or the [±]RNA class of DI particles) of VSV provides, through the subsequent action of that interferon, a common mechanism for the sparing of cells, i.e., the persistence of normally susceptible cells in the presence of lethal virus. The critical role we propose for interferon-mediated cell sparing in persistent infection by VSV in interferon-competent cells predicts that specific anti-interferon serum will shift the equilibrium between cell sparing ⇆ cell killing, and virus inhibition ⇆ virus replication, and precipitate a “crisis”, i.e., cell death and termination of persistent infection. Such results have been observed in the virus-cell systems thus far tested with anti-interferon serum.