Inhibition of Mengo virus by interferon

Gauntt, Charles J. (The University of Texas, Austin), and Royce Z. Lockart, Jr. Inhibition of Mengo virus by interferon. J. Bacteriol. 91:176-182. 1966.-The inhibition of Mengo virus replication in L cells resulting from interferon was studied quantitatively. Interferon was titrated on L cells with Western equine encephalomyelitis (WEE) virus as the challenge virus. One protective unit (PU) of interferon is the least amount of interferon which prevents cytopathic effects when a large multiplicity of WEE virus is added subsequent to overnight incubation with interferon. Ten PU of interferon reduced the yields of Mengo virus by about 90%. Larger doses of interferon, up to 220 PU, caused no further reduction in the amount of virus produced. Plaque formation by Mengo virus was also reduced in number by about 85 to 90%, but could not be further reduced. The plaques which formed on interferon-treated cells were reduced in size. We were unable to obtain a virus population with increased resistance to interferon action by use of five successive growth cycles in interferon-treated cultures. Analysis of the cell population for the proportion of cells able to act as infectious centers revealed that incubation of cells with 10 PU of interferon decreased the proportion of virus-yielding cells by 80%. The yield of virus per virus-producing cell was decreased by 40 to 60%. Despite the reduction in yields, plaques, and infectious centers resulting from interferon, all doses of interferon failed to prevent the complete destruction of the cells. Experiments with puromycin indicated that the cytopathic effects observed in L cells infected with Mengo virus required that a virus-directed protein be synthesized between 4 and 5 hr postinfection. The evidence suggested, therefore, that the Mengo virus genome was able to code for new protein synthesis in the absence of the production of infectious virus.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

The Identity of “Chromium Malate”

Recently, several studies on the effects of a compound named “chromium malate,” with the proposed formula “Cr₂malate₃·xH₂O” where x = 3.5 or 5, on the health of healthy and diabetic rats have appeared. However, the compound is poorly characterized, and knowing the identity of this material could be important in the interpretation of the previous and of future studies on the effects of this compound in animals. Consequently, the synthesis, characterization, and identity of this material were explored. A combination of spectroscopic, magnetic, and elemental analyses and mass spectral studies reveal that the compound is probably a polymer, not a discrete molecule, and does not have the composition previously reported. The repeating unit of the polymer possesses an antiferromagnetically coupled trinuclear Cr(III) core. The current study suggests that previous reports on chromium malate and its effects in animals must be viewed with caution.

     

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.     

Features independent of stain intensity for evaluating Feulgen-stained cells

Eleven features that are independent of stain intensity are described. Values greater than 1.4 times the average optical density were shown to define the visually darker areas of the image, which were considered to be condensed chromatin. Use of the 11 features permitted the discrimination between (1) lymphocytes and macrophages of rats, (2) macrophages from the spleens of rats and monocytes from peripheral blood and (3) macrophages from the spleens of rats injected with complete Freund's adjuvant and those from the spleens of normal rats.     

Relative Antiviral Resistance Induced in Homologous and Heterologous Cells by Cross-Reacting Interferons

Human cells incubated with human interferon become more resistant to vesicular stomatitis virus (VSV) than to Semliki Forest virus (SFV); monkey cells treated with monkey interferon become more resistant to SFV than to VSV. However, monkey cells incubated with human interferon developed relative antiviral activity identical to that induced by homologous interferon, and human cells developed characteristic human interferon-induced relative antiviral activity when exposed to monkey interferon. Therefore, cross-reacting interferons induce the relative antiviral activity characteristic of the interferon-treated cell rather than the cell of the interferon's origin. This relationship supports the hypothesis that interferon is not itself antiviral but rather induces cells to develop their own antiviral activity.     

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.     

Analysis of Additional Interference Occurring After the Removal of Interferon

Cultures of chick cells exposed to interferon continued to decrease in virus-producing ability during incubation after the interferon was removed. The rate of development of the additional interference and the degree of viral interference finally manifested were dependent on the concentration of interferon to which the cultures were exposed and the time of exposure. Additional interference occurred also in infected cells. Additional interference was inhibited by actinomycin D and puromycin. The best explanation of additional interference is that it results from interferon that is fixed to the cells during their initial period of contact.     

Replication of Western Equine Encephalomyelitis Virus: I. Some Chemical and Physical Characteristics of Viral Ribonucleic Acid 1

The ribonucleic acid (RNA) from Western equine encephalomyelitis (WEE) virions sedimented through sucrose gradients with a sedimentation coefficient of 40S. Another viral RNA which was always associated with infected cells possessed a sedimentation coefficient of 26S. Both 40S and 26S RNA had identical base compositions and densities. The 40S RNA displayed a hyperchromic effect when heated with a T(m) of 57.5 C. When 40S RNA was heated at 90 C and cooled rapidly, it sedimented with a coefficient of 26S. Dialysis of 40S RNA against distilled water changed its sedimentation coefficient to 26S. The presence of 8 m urea or 50% dimethyl sulfoxide in the gradients also altered the sedimentation rate of 40S RNA to 26S. In the latter case, the 26S RNA retained 10% of the infectivity originally added as 40S RNA. Dialysis of 26S RNA against 0.5 m NaCl or 0.05 m acetate buffer at pH 4.0 altered it so that about 50% of the radioactivity sedimented with a coefficient of 40S. Chromatography on methylated albumin-kieselguhr columns failed to separate 40S RNA from 26S RNA. Viral RNA either exists in two conformations which sediment differently in sucrose or contains an extremely labile portion near the center and is easily broken into two equal pieces.     

Interferon Induction by Viruses

Interferons are proteins of cellular origin capable of conferring virus resistance to vertebrate cells. Most cells do not produce interferons except in response to proper stimulation. Clearly, the stimulation of interferon production encompasses two phenomena. When stimulated, some cell systems produce their interferons by synthesizing new proteins. Other cell systems do not require the synthesis of new proteins to produce interferons, and still other cell systems may produce interferons by both means. Before much can be learned from the detailed study of the nature of the molecules which stimulate interferons, the type of phenomenon which the stimulus induces must be identified. Chick embryo tissues apparently make interferons by synthesizing new proteins. Many viruses stimulate interferon production in chick embryo tissues. Data available suggest that neither the protein nor nucleic acid moieties of the added virions act as inducing molecules. Also, double-stranded replicative form is probably not responsible. It is suggested that the inducer molecule may be cellular in nature and may be produced in response to a wide variety of insults among which are viral infections.