Optimization of the Interferon Assay Using Inhibition of Semliki Forest Virus-Ribonucleic Acid Synthesis

The method for assaying chicken interferon by its inhibition of viral ribonucleic acid (RNA) synthesis was optimized for the chicken embryo fibroblast-Semliki Forest virus (SFV) system, with respect to time, multiplicity of infection, and addition of actinomycin D and (3)H-uridine. Incorporation of (3)H into viral RNA is reproducible, and amounts to 10(4) to 2 x 10(4) counts per min per uninhibited culture per 1 muCi per 6 hr. The assay may be carried out in less than 1 day and is sensitive (0.05 units/ml) and exact (+/-20% of the mean titers on different days); it can be used for purification procedures as well.     

Action of Interferon: Kinetics and Differential Effects on Viral Functions

A highly purified rabbit interferon was tested for its capacity to inhibit various manifestations of infection of primary rabbit kidney (RK) cells with vesicular stomatitis (VS) virus. A kinetic analysis of the actinomycin-sensitive phase of interferon-induced cellular resistance revealed that RK cells could transcribe virtually all of the hypothetical antiviral messenger ribonucleic acid (mRNA) within 3 hr. Similar exposure to interferon reduced virus yield by 95 to 99% and markedly inhibited cytopathic effect on RK cells infected at a multiplicity of 10 or less. Interferon was less effective in blocking cytopathic effects when RK cells were infected at a multiplicity of 100. However, RK cells pretreated with the same amount of interferon and infected at a multiplicity of 100 failed to incorporate (3)H-amino acids into structural or nonstructural proteins of VS virus identified by polyacrylamide gel electrophoresis. Despite this inhibition of viral protein synthesis, interferon did not prevent the switch off by VS virus of cellular protein synthesis. The rapidity with which a high multiplicity of VS virus switched off cellular protein synthesis, even in cells rendered resistant to viral infection by interferon, is further evidence that this reaction is caused by an infecting virion component rather than by a newly synthesized viral product.     

Interferon Production and Inhibition of Host Synthesis in Cells Infected with Vesicular Stomatitis Virus

Infection of L cells with wild-type (L(1)) vesicular stomatitis virus at high or low multiplicities does not result in the production of interferon; however, infection of L cells with low multiplicities of a small-plaque mutant (S(2)) results in the synthesis of large amounts of interferon. In chick embryo (CE) cells, both viruses induce synthesis of interferon; there is no significant multiplicity effect in CE cells. The rate and efficiency of shutoff of macromolecular synthesis in the different host cells is a critical factor in determining the ability of the viruses to induce interferon synthesis. If host ribonucleic acid or protein synthesis is shut off by the virus before the required new ribonucleic acid is transcribed or translated, interferon production does not occur. The relative yield of the two viruses in CE and L cells is not related to the effects of interferon produced during the course of infection.     

Isolation and Characterization of a Double-Stranded Ribonucleic Acid from Penicillium chrysogenum

Double-stranded ribonucleic acid has been obtained from cells of the fungus Penicillium chrysogenum. This ribonucleic acid appears to be associated with mycophage and is an efficient inducer of interferon Its. extraction and partial purification are discussed, and evidence for its double-stranded and ribosidal nature is reviewed. The implications of viral nucleic acid in the life processes of fungi are considered.     

Effect of colchicine on virus-induced interferon synthesis in chick embryo cells

Colchicine, at a concentration of 5 X 10(-4) M (0.2 microgram/ml), inhibits interferon synthesis induced by theLee strain of influenza B virus in chick embryo cells, but it does not influence the release of preformed interferon from cells. The same drug concentration does not affect the overall synthesis of cellular RNA and protein. The inhibition of interferon synthesis by colchicine is a temperature-dependent process and is not manifested at 0 degrees C. Colchicine is found to be most effective when it is introduced into the medium at early stages of infection. It is suggested that colchicine inhibits the formation of messenger RNA for interferonogenesis.     

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.     

Destruction of L Cells by Mengo Virus: Use of Interferon to Study the Mechanism

Interferon, when added to L cells, inhibited the synthesis of infectious Mengo viral ribonucleic acid, hemagglutinins, and infectious virus by 85 to 95%. Serum-blocking antigens were also reduced by the action of interferon, but threefold excess amounts of these antigens accumulated in interferon-treated cultures above the amounts expected for the quantity of infectious virus that was produced in these cultures. Radioautographic analysis showed that 28 to 36% of the cells of an interferon-treated population synthesized viral ribonucleic acid and 36 to 47% produced viral antigens as determined by an immunofluorescence technique. Despite the reductions in synthesis of viral components, all cells in an interferon-treated culture underwent cytopathic effects at the same time as cells in infected cultures which had not been treated with interferon. The results are compatible with the hypothesis that the cell destruction which results from the infection of L cells with Mengo virus is due to a protein which is coded for by the virus but is not a component of the mature virion.     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

Temperature-sensitive Steps in the Biosynthesis of Venezuelan Equine Encephalitis Virus

In contrast to Eastern equine encephalitis virus, the replication of Venezuelan equine encephalitis (VEE) virus was strongly inhibited at 44 C in chick embryo cells. The inhibited steps were analyzed by shifting the incubating temperatures up or down, and by determining during the shifts the rate and extent of infectious ribonucleic acid (RNA) synthesis, intact virus synthesis, and formation of complement-fixing antigen or of antigen detectable by a direct fluorescent-antibody technique. The inhibition appeared to be due to two temperature-sensitive steps involved in the synthesis of VEE virus in chick embryo cells. The first step of inhibition at 44 C occurred early in virus replication and could be completely reversed simply by transferring cultures to 37 C. The inhibition appeared to take place at some point between the time when the virus entered the cell and was uncoated and the beginning of viral RNA synthesis. The second temperature-sensitive step in VEE virus synthesis was irreversible; it occurred at a point after the synthesis of viral RNA, and before the formation of virus protein measured as complement-fixing antigen or as antigen that could be stained with fluorescent antibody.     

Effect of Infection with Ribonucleic Acid Bacteriophage R23 on the Inducible Synthesis of β-Galactosidase in Escherichia coli

Infection by ribonucleic acid (RNA) bacteriophage R23 inhibited the synthesis of beta-galactosidase in Escherichia coli. The inhibition, although not complete, was apparent shortly after infection and was maximal after the first 20 min of infection. R23 diminished the beta-galactosidase-synthesizing capacity when inducer was added after phage infection, but not when infection followed inducer removal. These findings suggested that the primary effect of R23 on enzyme-forming capacity was limitation of synthesis of enzyme-specific messenger RNA. Studies with ultraviolet irradiated phage and amber mutants of R23 indicated that the inhibitory process could be separated into two phases. Early inhibition did not require the expression of the viral genome, whereas late inhibition required the expression of the viral RNA synthetase cistron.     

Establishment and Maintenance of the Interferon-Induced Antiviral State: Studies in Enucleated Cells

Requirements for the physical presence of the cell's nucleus for the establishment and maintenance of the interferon-induced antiviral state were investigated. Enucleated chicken embryo fibroblasts were obtained by cytochalasin B treatment during centrifugation. The inhibition of vaccinia virus cytoplasmic DNA synthesis, monitored by autoradiography, was used to measure the antiviral activity resulting from interferon treatment. The antiviral state is not established in cells treated with interferon after removal of their nuclei. On the other hand, cells first treated with interferon for 6 or 12 h and then enucleated express the antiviral state. Furthermore, the antiviral state is maintained in enucleated cells for 16 h after enucleation. The antiviral state appears to be more stable in enucleates than in the residual nucleated cells found in the same cultures. Single cells of antiviral populations are found to be either fully permissive or fully restrictive to vaccinia DNA synthesis. The effect of an increasing intracellular multiplicity of infectious virus is to overcome the antiviral cell's block against viral DNA synthesis.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.     

Metabolic Properties of Early and Late Vaccinia Virus Messenger Ribonucleic Acid

In vaccinia-infected cells, 60% of the viral messenger ribonucleic acid (mRNA) was associated with polyribosomes, and the remainder sedimented in a broad peak in the 30 to 74S region. The quantity of mRNA in polyribosomes was sharply reduced late in the infectious cycle [9 hr postinfection (PI)] to less than 30% of the 2-hr value. However, protein synthesis proceeded at a nearly constant rate from 2 to 13 hr PI. This ability of small quantities of late mRNA to support as much protein synthesis as do the much larger quantities of early mRNA was not due to an increase in stability, since late mRNA decays with a half-life of 13 min, whereas early mRNA has a half-life of 120 min. A similar decrease in viral mRNA synthesis without an accompanying decrease in viral protein synthesis was observed when deoxyribonucleic acid synthesis is inhibited. In contrast to the rapid decay of the late mRNA which was present in polyribosomes, the mRNA which sedimented in the 30 to 74S region remained unchanged even after a 2-hr period of exposure to actinomycin. The rate at which infected cells lose the capacity to synthesize specific viral proteins after exposure to actinomycin D was consistent with the half-life values of early and late mRNA that were observed.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Phospholipid Synthesis in Sindbis Virus-Infected Cells

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.