Igor Tamm

Characterization and Separation of an Inhibitor of Viral Hemagglutination Present in Urine

Summary Normal human urine contains a highly active inhibitor of viral hemagglutination, effective against PR8, Lee, FM1, swine, mumps, NDV, and GDVII viruses but inactive against PVM. The inhibitor concentration may approach 2 mg%. Considerable amounts of purified material, active at 0.0001 μg/cc, can be obtained readily. The activity of the inhibitor as it occurs in urine is inversely related to the salt concentration; it should be emphasized that in NaCl solutions of moderate strength it precipitates.

A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1.

Earlier studies demonstrated the induction of beta 2-interferon (IFN-beta 2) in human diploid fibroblasts (FS-4 strain) exposed to tumor necrosis factor (TNF). These studies suggested that IFN-beta 2 mediates an antiviral effect in TNF-treated cells and exerts a feedback inhibition of the mitogenic effect of TNF. Here we demonstrate that the expression of the antiviral action of TNF can be enhanced by prior exposure of FS-4 cells to trace amounts of IFN-beta 1. IFN-beta 1, at a higher concentration, can directly increase the expression of IFN-beta 2. Exposure of cells to TNF enhanced IFN-beta 2 (but not IFN-beta 1) mRNA expression in response to poly(I).poly(C), an IFN inducer which is also known to stimulate FS-4 cell growth. Platelet-derived growth factor and interleukin-1 also led to the increased expression of IFN-beta 2. However, platelet-derived growth factor and interleukin-1 could override the antiviral effect of TNF and also that of exogenously added IFN-beta 1. Our data suggest that a complex network of interactions that involves the endogenous production of IFN-beta 2 is triggered by several growth-modulatory cytokines. Cellular homeostasis is likely to represent a balance between the induction of IFN-beta 2 by these cytokines and their ability to override the inhibitory actions of IFN-beta 2.

STUDIES ON THE MECHANISM OF POLIOVIRUS-INDUCED CELL DAMAGE. I. THE RELATION BETWEEN POLIOVIRUS,-INDUCED METABOLIC AND MORPHOLOGICAL ALTERATIONS IN CULTURED CELLS.

Abstract Infection with the attenuated strain of poliovirus type 2 depressed cellular RNA and protein synthesis in parallel both in human embryonic lung cells (diploid) and in ERK cells. In HeLa cells protein synthesis was depressed more rapidly than RNA synthesis. In all experiments an input multiplicity of 100 PFU/cell was used. Guanidine, at a concentration of 1110 μ M , completely prevented production of new virus in human embryonic lung cells, but had only a slight delaying effect on the virus-induced inhibitions of RNA and protein synthesis. Guanidine did markedly delay the development of morphological changes in infected cells. Twenty-one hours after infection most of the drug-treated cells appeared morphologically normal, although the rates of cellular RNA and protein synthesis in these cells were as much depressed as in the untreated cells at 5–6 hours, at which time the untreated cells were already undergoing advanced morphological changes. The simplest hypothesis to explain these findings is that the virus-induced metabolic depressions are not the sole, immediate, and direct cause of the morphological changes, which in untreated infected cells accompany, or follow shortly after, the active replication of virus. However, the eventual degeneration of guanidine-treated infected cells, which occurs in the absence of virus multiplication, may indeed be due to the virus-induced inhibitions in cellular metabolism.

The role of cytoplasmic membranes in poliovirus biosynthesis

Abstract Chemical analysis and electron microscopy show that isopycnic centrifugation in discontinuous sucrose density gradients separates smooth and rough cytoplasmic membranes of HeLa cells. By 3.25 hours after poliovirus infection, there is evidence of marked proliferation of cytoplasmic membranes in the smooth microsomal fraction. Sixty-five percent of labeled virus-specific RNA is found in this fraction after a 2.5-min pulse of uridine-3H 3.25 hours after infection; however, after a 10-min pulse, most of the labeled RNA is found in the rough microsomal fraction. Over 50% of newly synthesized viral protein is found in the rough microsomal fraction throughout a 30-min chase period following a 3-min pulse of leucine-3H. This pattern of distribution of viral RNA and protein is dependent on the presence of cytoplasmic membranes. The major site of translation of viral RNA is associated with rough membranes, whereas replication of viral RNA takes place in association with smooth membranes. Results after addition of guanidine to infected cells support the hypothesis that viral RNA synthesis is initiated in a complex associated with smooth membranes.

Replication of Semliki Forest virus: an electron microscopic study.

Abstract The growth of Semliki Forest virus was studied with the electron microscope during a single cycle of viral replication in chick embryo cells. The spherical virus particle consists of a nucleoid closely wrapped in a membrane covered by projections. After a latent period of 2–3 hours, infectious virus is produced at a nearly constant rate of approximately 200 PFU/cell per hour until the ninth hour. During the period of rapid production of virus, individual free virus nucleoids are found scattered in the cytoplasmic matrix. The nucleoids appear to migrate singly to the plasma membrane, where complete virus particles are formed by a budding process. An envelope, consisting of a portion of the membrane covered with projections, encloses a nucleoid as it is extruded into the extracellular space. Few complete virus particles are found within cytoplasmic vacuoles. After 9 hours, the rate of virus production decreases, and few developing virus particles are seen. Cells begin to show marked cytopathic changes, including vacuolization and condensation of the cytoplasm. In addition to being scattered singly in the cytoplasmic matrix, nucleoids aggregate around vacuoles and in paracrystalline arrays. Such accumulations of nucleoids do not appear to have a functional role in the production of Semliki Forest virus particles.

The inhibition of DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells

Abstract 5, 6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB; 75 μM) inhibited only 60–75% of nuclear heterogeneous RNA (hnRNA) synthesis in HeLa cells, while the appearance of labeled cytoplasmic poly(A)-containing messenger RNA (mRNA) was reduced by approximately 95%. DRB-sensitive and resistant transcripts spanned the entire size range of hnRNA molecules. The relative poly(A) content of DRB-resistant hnRNA molecules was the same as that of normal hnRNA. In contrast to the effects of 3′deoxyadenosine, DRB appeared to inhibit the initiation of hnRNA chains, but did not directly interfere with labeling of poly(A). We present an interpretation of these results and propose how the results might be used to gain more information about nuclear RNA metabolism.

Reovirus Type 3: Physical Characteristics and Interaction with L Cells.

Abstract The physical characteristics of reovirus type 3 and its interaction with L cells have been investigated. After repeated passage of the Dearing strain in L cells, a variant emerged which was characterized by decreased sensitivity to specific antibody and by diminished release from cells. The original and variant strains were not significantly different in their antigenic constitution, size, or fine structure. The amount of each which adsorbed to L cells in 2 hours was approximately 80% of that adsorbed in 4 hours. The latent period was 7 hours and the maximal yields were reached at 15 hours with both the original and the variant strains. Both caused the development of a perinuclear inclusion in infected cells. The inclusion was Feulgen negative; it contained viral antigenic material. Both strains were insensitive to inhibition by 5-fluoro-2′-deoxyuridine and 5-bromo-2′-deoxyuridine, whereas vaccinia virus was markedly inhibited. Mitomycin C and actinomycin D inhibited reovirus to a lesser extent than vaccinia virus under most conditions used. However, after pretreatment of cells with actinomycin D, their ability to produce reo or vaccinia viruses was restricted to a similar degree. 5,6-Dibromo-1-β- d -ribofuranosylbenzimidazole and aminopterin inhibited the original Dearing strain and vaccinia virus to a similar degree. The variant strain was slightly less sensitive to the inhibitors than the original. The nucleic acid of reovirus 3 appears to be of the ribose type.

Halobenzimidazole Ribosides and RNA Synthesis of Cells and Viruses

Publisher Summary This chapter brings together in a systematic way the recent work as well as some of the earlier studies on the mode of action of halogenated ribofuranosylbenzimidazoles on cellular and viral biosynthesis. Investigations of the cellular and viral biosynthesis, in which these compounds have been used as exploratory tools are also reviewed in this chapter. Compounds in this class of benzimidazoles possess the unique biological activity of selective inhibition of the synthesis of nuclear heterogeneous RNA (hnRNA) in cells of cold-blooded animals, including insects and in cells of avian and mammalian species. Because of the universality of the synthesis of hnRNA in animal species and because a portion of hnRNA sequences becomes mRNA, the action of halobenzimidazole ribosides on cellular biosynthesis is of broad interest. The action of these derivatives is also relevant to the process of transcription of the viral genomes of certain RNA and DNA viruses. In addition, this chapter briefly discusses three groups of structurally distinct bcnzimidazolcs that possess biological actions entirely different from those of halobenzimidazole ribosides: 2-(α-hydroxybenzyl) benzimidazole and certain related compounds are selective inhibitors of picornavirus multiplication; 5-methyl-2-D-ribobenzimidazole and congeners and 5-hydroxy-l-methylbenzimidazole and certain related compounds restore the capacity of chorioallantoic membranes from older chicken embryos to produce influenza virus in high yield.

Interferon effects on the growth and division of human fibroblasts.

Abstract The overall rate of proliferation of human fibroblasts in culture is reduced at interferon concentrations greater than 40 international reference units (U)/ml. Inhibition is near maximal at 640 U/ml, at which concentration the doubling time between 24 and 72 h after beginning of treatment is increased 2–3 times over the control value. Inhibition of cell proliferation was not readily reversible upon removal of interferon and refeeding of cultures. Study of the mitotic behavior of individual cells showed that the first intermitotic interval after beginning of treatment with interferon (640 U/ml) was prolonged in about two-thirds of the cells. In this fraction, many cells failed to divide again after the second post-treatment mitosis, while others exhibited a progressively increasing intermitotic interval with subsequent divisions. One-third of the interferon-treated fibroblasts initially divided at a rate similar to the rate of proliferation of control cells, but subsequently these cells also slowed down and finally stopped dividing. After treatment at 640 U/ml for 3 days, the rates of DNA, RNA, and protein synthesis were depressed to 86, 75, and 64% of control values, respectively. However, the interferon-treated fibroblasts had grown larger than control cells as indicated by the following parameters: cell attachment area, 165%; volume, 131%; DNA content, 130% and protein content, 150%. Thus, interferon does not prevent cell growth, but interferes with cell division.

Membranous Structures Associated with Translation and Transcription of Poliovirus RNA

Poliovirus RNA and proteins are synthesized in association with distinct membranous structures that were separated by means of Isopycnic centrifugation of cytoplasmic extracts in discontinuous sucrose-density gradients. Viral RNA is replicated in a structure that contains rapidly labeled replicative intermediate RNA and viral RNA polymerase associated with the smooth membrane fraction. In sucrose gradients this viral RNA replication complex is distributed at densities in the range of 1.12 to 1.18 grams per cubic centimeter. Viral proteins are synthesized on polyribosomes bound to membranes and sediment with polyribosomes at densities of less than 1.25 grams per cubic centimeter.