Hilton B. Levy

Inhibition of biologic activity of poly I: poly C by human plasma.

Summary Human serum and plasma have been shown to abolish the pyrogenicity of poly I:poly C. Chicken serum and, to a lesser extent, fetal calf serum also possess this inhibitory effect, while serums of six other animal species do not alter the pyrogenic properties of this compound. The inhibitory effects are time- and temperature-dependent and suggest that they are enzymatic in origin.

Studies on the mechanism of action of interferon

Abstract An investigation into the biochemical action of interferon has been carried out. When uninfected chick embryo fibroblast tissue culture cells were exposed for 18–24 hours to interferon at levels 10 times that which would cause a 50% inhibition in virus growth, no regular effects were found on the rate of synthesis of protein, DNA, rapidly labeled RNA, or of a number of smaller molecules. In CEF cells infected with Sindbis virus, on the other hand, interferon inhibited the synthesis of rapidly labeled, phenol-released RNA, both early in the course of infection, when little or no virus RNA was being made, and later. In other experiments, a 15-minute pulse exposure of cells to radioactive uridine was followed by a cold uridine “chase,” and slowly labeled RNA was studied. Pretreatment of the cells with interferon led to a decreased amount of radioactivity in the slowly labeled RNA in infected and uninfected cells. It is tentatively suggested that this latter effect is due to an inhibition of transfer from rapidly labeled to slowly labeled RNA.Abstract An investigation into the biochemical action of interferon has been carried out. When uninfected chick embryo fibroblast tissue culture cells were exposed for 18–24 hours to interferon at levels 10 times that which would cause a 50% inhibition in virus growth, no regular effects were found on the rate of synthesis of protein, DNA, rapidly labeled RNA, or of a number of smaller molecules. In CEF cells infected with Sindbis virus, on the other hand, interferon inhibited the synthesis of rapidly labeled, phenol-released RNA, both early in the course of infection, when little or no virus RNA was being made, and later. In other experiments, a 15-minute pulse exposure of cells to radioactive uridine was followed by a cold uridine “chase,” and slowly labeled RNA was studied. Pretreatment of the cells with interferon led to a decreased amount of radioactivity in the slowly labeled RNA in infected and uninfected cells. It is tentatively suggested that this latter effect is due to an inhibition of transfer from rapidly labeled to slowly labeled RNA.

Molecular basis of the action of interferon

Abstract The interaction of labeled Mengo virus particles with mouse L cells has been studied at very early times after infection. At 30 minutes after infection, a 50 s particle bearing intact viral RNA has been isolated from the cytoplasm of the normally infected cell. This 50 s particle is possibly a complex of viral RNA with the 40 s ribosomal subunit. By study at different intervals after infection, its formation can be related to the assembly of a functional polysome which sediments at 240 s. Viral proteins, including the viral RNA polymerase, are detectable on this complex, although it is not certain that they are synthesized there. Newly formed viral RNA molecules are also processed through the 50 s particle into a similar 240 s polysome. In cells exposed to interferon before infection, the 50 s particle bearing viral RNA is not detected, although a 240 s complex is assembled late in infection. However, this complex does not translate the viral genome to a significant extent, as reflected in a greater than 90% reduction in the synthesis of the viral RNA polymerase and progeny RNA molecules. Although other possibilities are not excluded, the interaction of viral RNA with the smaller ribosomal subunit may be a necessary step in the formation of a functional polysome. The prevention of this interaction may explain the antiviral action of interferon.

Ribosomes: Effect of Interferon on Their Interacton with Rapidly Labeled Cellular and Viral RNA's

Rapidly labeled RNA of mouse L cells and labeled RNA of Mengo virus, unlike cellular RNA labeled under steady-state conditions, form detectable complexes with L-cell ribosomes. These ribosome-RNA complexes formed in vitro appear analogous to those assembled during polysome formation in vivo. When ribosomes are prepared from L cells exposed to homologous interferon, their capacity to associate with cell messenger is preserved, while their ability to interact with viral RNA is markedly reduced. The ribosomes from cells exposed to interferon are thus altered selectively to permit only certain messages to be bound and translated.

Liposome-mediated immunotherapy against respiratory influenza virus infection using double-stranded RNA poly ICLC

The use of liposome delivery technology to enhance the antiviral activity of poly ICLC (an immunomodulating dsRNA) while decreasing its intrinsic toxicity is evaluated in this study. The antiviral efficacies of free and liposome-encapsulated poly ICLC were evaluated and compared using a lethal respiratory influenza A virus infection in mice. The toxicity profiles of free and liposome-encapsulated poly ICLC were compared by determining the extent of hypothermia and loss in body weights in mice pretreated with these drugs. Poly ICLC was encapsulated in cationic liposomes prepared by the freeze drying method. To determine the antiviral efficacies of free and liposome-encapsulated poly ICLC, mice were intranasally pretreated with two doses of poly ICLC (free or liposomal, 1 mg/kg/dose) given 48 h apart. At various times post pretreatment, mice were intranasally challenged with 10 LD50 mouse-adapted influenza A/PR/8 (H1N1) virus. The survival rates of the mice were determined at day 14 post infected and compared to the untreated control mice. Results indicate mice pretreated with liposome-encapsulated poly ICLC within 3 weeks prior to virus challenge were completely protected (100% survival compared to 0% for the untreated control group, p < 0.001), while window of protection provided by free unencapsulated poly ICLC was 12 days. When the toxicity profiles of free and liposome-encapsulated poly ICLC were compared, it was found that hypothermia and body weight loss induced by poly ICLC were either completely mitigated or significantly reduced in mice given equivalent doses of poly ICLC in the liposome-encapsulated form. These results suggest that liposomes are an excellent drug carrier for poly ICLC, that liposome-encapsulated poly ICLC may provide a safe and effective immunotherapeutic approach for the prevention of respiratory influenza virus infections.

Some Factors Affecting the Interferon-Induced Antiviral State

Summary and Conclusions The results of the present study indicate that several factors affect the interferon-induced resistance to viruses in tissue culture. Reaction of cells and interferon results in rapidly increasing antiviral activity over about 7 hours. Thereafter antiviral activity remains relatively stable in the presence of an unchanging amount of interferon in the extracellular fluid. Continued presence of the initial concentration of interferon was required to maintain the established level of resistance. Addition of more interferon resulted in a further rise of resistance. The level of cellular antiviral activity was determined mainly by the concentration of interferon rather than by the amount applied. Maintenance of resistance required continued cellular protein synthesis and also probably required continued cellular RNA synthesis. These findings support the previous proposal that development of antiviral activity by cells exposed to interferon is due to the induction of a cellular antiviral protein. The results also suggest that maintenance of stable antiviral activity in the presence of interferon could be the result of continued induction by interferon of enough antiviral protein to offset decay.

Effect of Interferon on Early Interferon Production.

Chick embryo cells given prior treatment with interferon make new interferon earlier and in larger quantities upon stimulation with Chikungunya virus than cells not so treated. By the criterion of loss of sensitivity to actinomycin, the time needed for formation of messenger RNA for interferon was decreased in the primed cells. Thus interferon affects virus action within 1 hour after infection.

Studies on the Mechanism of Interferon Action. II. The Effect of Interferon on Some Early Events in Mengo Virus Infection in L Cells.

Abstract The effect of interferon on early events of Mengo virus replication in suspended cultures of L cells has been studied. Interferon, at levels that almost completely block the production of Mengo virus, has no ameliorative effect on the early inhibition of normal cell protein synthesis induced by Mengo virus. Such infected interferon-treated cells go on to die in a manner similar to infected cells not treated with interferon. However, in the presence of interferon, the rapid inhibition of normal cell RNA synthesis seen in Mengo virus infection does not appear until about an hour later than usual.

Studies on Poly I:C toxicity in experimental animals☆☆☆

Abstract Polyinosinic:polycytidylic acid copolymer (Poly I:C), a synthetic double stranded RNA, has been proposed as a candidate for clinical introduction as an antitumor agent. In preclinical toxicologic evaluation, acute lethality studies were undertaken in 3 mouse strains, anaphylaxis studies in guinea pigs, and acute and subchronic toxicities were evaluated in rhesus monkeys and beagle dogs following daily parenteral dosing for up to 28 days. Poly I:C is more lethal to mice following iv administration than ip, and there appear to be differences in mouse strain susceptibility. No evidence of anaphylaxis was seen in guinea pigs. Signs of toxicity in dogs and monkeys were dose related, were qualitatively similar in the 2 species, and included hypoactivity, anorexia, emesis, diarrhea, ataxia and weight loss. Clinical changes included anemia, elevations of transaminases, lactic dehydrogenase, and alkaline phosphatase, decreased clotting rate and increased prothrombin time. Pathologic changes consisted of focal hemorrhages, infarction or congestion of a variety of organs, vasculitis and decreased erythroid hematopoiesis. The effects were more severe in dogs than in monkeys for equivalent mg/kg doses. In the monkey iv administration produced greater toxicity than im or ip administration. Signs of toxicity and pathologic changes appeared to be reversible in both dogs and monkeys after cessation of dosing.

Intracellular sites of poliovirus reproduction

Abstract A study of the early intracellular events in poliovirus reproduction has been made using fluorescent antibody staining and high resolution autoradiography with tritiumlaleled protein and nucleic acid precursors. Within an hour after infection there was increased nucleolar RNA turnover as determined by uptake of tritiated H3-uridine and H3-cytidine. This stimulated turnover continued for 3 1 2 –4 hours , then the uptake of these compounds markedly declined. Thymidine incorporation into DNA was affected little or not at all until nucleolar RNA metabolism declined and at the same time DNA metabolism also declined rapidly. Total cellular protein synthesis was retarded by 1 1 2 hours after infection, as revealed by H3-histidine uptake. However, at about 2 hours after infection there appeared in the cytoplasm an antigen that stained weakly in a specific fluorescent antibody staining test. At 2–3 1 2 hours after infection, there were a large number of cells showing fairly intense specific fluorescence in the nonnucleolar portion of the nucleus. The nucleoli themselves were unstained, but were surrounded by bright rings of fluorescence. By 4–5 hours after infection an increasing number of cells showed bright cytoplasmic particulate fluorescence. This was about the time when parallel virus growth studies revealed the first presence of new intracellular virus.