The virion of mengovirus contains anti-interferon activity.

Many viruses are resistant to the antiviral state induced by interferon (IFN) in certain cell lines because they produce factors having anti-IFN activity (AIA). We show here that is+, a mengovirus strain resistant to low concentrations of IFN, also produces an AIA, but that an IFN-sensitive mutant (is-1) does not. This activity was detected when is+ rescued is-1 from the antiviral state induced in mouse L cells by mouse IFN. The mengovirus AIA was found in cell lysates prepared from cells infected with is+, and in the virions of purified, inactivated is+ but not in lysates prepared from is-1-infected cells or in the is-1 virion. Also the pentameric subunits that make up the viral capsid contained AIA, whereas the individual monomeric subunits that constitute the pentamers did not. We also describe an assay system for detecting and quantitating the mengovirus AIA.

pH 2 instability of a mutant of mengovirus is related to its interferon sensitivity.

The is-1 mutant of mengovirus is 100 times more sensitive to interferon (IFN) than wild type, as measured by a yield reduction assay in the G3 line of mouse L cells, and is also much more readily inactivated at pH 2. Neither isolated nor encapsidated RNA is degraded under these conditions, which suggests that the pH-sensitive region resides on the virus coat. One-third of the viruses selected for resistance to low pH also showed enhanced resistance to IFN. Attempts to isolate IFN resistant strains directly from is-1 stocks were unsuccessful. These results suggest that either a capsid protein or its precursor is an active anti-IFN agent.

Effects of the copper chelators diethyldithiocarbamate and bathocuproine sulfonate on interferon and its antiviral state.

Treatment of mouse L cells with two structurally unrelated chelators of copper ions, diethyldithiocarbamate (DDC) or bathocuproine sulfonate (BCS), completely inhibited the ability of interferon (IFN) to inhibit mengovirus growth. However, mengovirus virions were inactivated by incubation with DDC, and DDC induced a generalized inhibition of cellular RNA and protein synthesis, possibly combined with the inactivation of one or more specific enzymatic activities involved in the establishment or maintenance of the antiviral state. In contrast, BCS had no effect on either cell or virus growth. BCS combined with trace copper ions in the growth medium and the resulting complex prevented IFN from interacting properly with cellular receptors, apparently by binding noncovalently to the IFN molecules. In some cases, IFN was irreversibly inactivated, probably due to oxidation of essential cystein, tyrosine, or tryptophan residues by the (BCS)2Cu2+ complex. BCS was at least as effective as anti-IFN antibody at neutralizing cell-bound IFN, and has a number of advantages over it.

Selection and characterization of interferon response mutants from mouse L929 fibroblast cells.

Interferon (IFN) response mutants were selected from mouse L929 fibroblast cells and their specific resistance to is-1, an IFN-sensitive mutant of mengovirus, was studied. The standard L cell subline used in our laboratory (G3), is resistant to is-1 infection after pretreatment with low levels of IFN. Two clonal sublines that support the growth of is-1 in the presence of IFN (AS-4 and TA-6) were isolated from it, and two revertant lines (AS-4R1 and TA-6R1) were subsequently selected from AS-4 and TA-6. The kinetics of is-1 growth in the presence of IFN were found to vary in each of these sublines. Specific resistance to is-1 cannot be accounted for by enhanced induction of IFN, ability to bind IFN, or increased 2'-5'-oligo(A)-dependent endonuclease activity. AS-4 and TA-6 appear to have arisen through loss of one or more whole chromosomes. The origin of TA-6R1 is unclear.

Selection and characterization of an interferon-responsive clonal cell line of HeLa cells.

HeLa cells generally do not respond well to interferon (IFN). We have used is-1, an IFN-sensitive mutant of mengovirus, to select a clone of IFN-responsive HeLa cells (F-H12). At moderate levels of human alpha/beta IFN, is-1 yields were fivefold lower in these cells than in similarly protected control cells. In contrast, wild-type mengovirus, vesicular stomatitis virus and a wild-type and thymidine kinase-negative strains of herpes simplex virus type 1 grew equally well in both cell lines. By a cell survival assay, the F-H12 line was up to 100 times more responsive to IFN than the parental line when challenged by is-1. 2'-5'-Oligo(A)-dependent endonuclease activity was the same in both lines. These observations cannot be accounted for by enhanced induction of IFN following infection.

Growth of wild-type and interferon-sensitive mengovirus in tk- L cells.

Cells lacking thymidine kinase (tk) have been reported to fail to respond to the action of interferon (IFN). While some laboratories have confirmed this observation, others have failed to do so. We studied the effect of IFN on four freshly isolated tk- lines of mouse L cells infected with mengovirus. In all cases, normal antiviral activity was induced. The antiproliferative activity of IFN was studied using the parental L cell line, a tk- derivative, and a tk- (tk+) subline into which the tk gene of herpes simplex virus was introduced. All three lines had a doubling time of about 20 h. In all cases, 2,000 U/ml of IFN increased this time to about 50 h. In contrast to the above results, an IFN-sensitive mutant of mengovirus (is-1) grew much better in protected tk- cells than in protected normal cells. This phenomenon appears to be dependent on the fact that tk- cells were routinely maintained in medium containing 5-bromodeoxyuridine (BUDR). In the absence of this drug, the virus behaved normally. The implications of this observation are discussed.

The role of interferon induction in the interferon sensitivity of the mengovirus mutant, is-1.

An interferon-sensitive mutant of mengovirus has been shown to specifically induce interferon in infected cells. Although this appears to account for the sensitivity to interferon observed by others in an L(Y) cell line, it cannot account for the even greater sensitivity observed in our G3 line of mouse L cells.

Antiviral activities directed against wild-type and interferon-sensitive mengovirus.

Two distinct antiviral activities can be detected in L cells treated with low levels of interferon and infected with a one-step interferon-sensitive mutant of mengovirus (is-1). The first antiviral activity (AVA-1) primarily delayed virus RNA and protein synthesis and thereby lengthened the virus replication cycle. It did not prevent cell death. The second antiviral activity (AVA-2) allowed the virus-induced inhibition of host macromolecular synthesis but inhibited all other virus functions. By 9 to 12 h post-infection host synthesis resumed and most cells survived. The data suggest that some step in the virus replication cycle activates AVA-2 leading to the destruction of the virus genome 6 to 12 h after infection. In unprotected cells the yields of parental virus (is+) and is-1 were similar. No qualitative or quantitative differences in virus products were observed by several techniques. The is-1 virus seems to have lost a wild-type function which normally blocks the action of AVA-2.

Studies on an interferon-sensitive mutant of mengovirus: effects on host RNA and protein syntheses.

Interferon induces an activity which strongly inhibits the growth of is-1, an interferon-response mutant of mengovirus. This activity is not expressed in protected cells infected with either is+ (the wild-type parent) or vaccinia virus, or in cells infected with is-1 in the presence of actinomycin D. A failure of is-1 to shut off host RNA and/or protein synthesis could explain these observations. The present paper, however, shows that is-1 and is+ are equally effective in suppressing host synthesis, and suggests that is+ actively inhibits the interferon-mediated activity directed against is-1.

Interferon production by individual L cells.

Using a protected centre technique in which agarose prevents the diffusion of interferon from individual producing cells, we have shown that essentially every cell in a monolayer of mouse L cells can be induced to produce interferon by infection with Newcastle disease virus (NDV). The amount of interferon produced by individual cells appeared to be highly variable, even when cloned cells and viruses were used. U.v.-irradiated virus lost its capacity to induce interferon in L cells and to infect chick embryo fibroblasts at the same rate. A small proportion of cells (1 X 10-6 to 10 X 10-6) appeared to produce interferon constitutively. This fraction was increased threefold by u.v. irradiation of the cells, and up to 10-fold by exposing cells to the mutagen ethyl methane sulphonate.

Photosensitization of herpes simplex virus type 1 with neutral red.

Commercial neutral red (NR) originally containing at least 8 components was purified by thin layer chromatography. Herpes simplex virus type 1 (HSV-1) treated in vitro with 30 microgram/ml of purified NR became sensitive to light inactivation within 2 min but rapidly lost this sensitivity upon dilution. Similarly, virus grown in the presence of NR lost its photosensitivity upon dilution of the virus stock. In both cases the kinetics of inactivation appeared to be multi-hit. Photoinactivation of intracellular virus was most effective when NR was applied between 6 and 12 h post-infection. The most efficient inactivation occurred when virus at pH 8.8 was irradiated by light at a wavelength of 470 nm.