The relative in vitro antiviral activities of three related nucleoside carboxamides, ribavirin (1-3-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), tiazofurin (2-3-D-ribofuranosylthiazole-4-carboxamide), and selenazole (2-p-D-ribofuranosylselenazole-4-carboxamide), were studied against selected DNA and RNA viruses. Although the activity of selenazole against different viruses varied, it was significantly more potent than ribavirin and tiazofurin against all tested representatives of the families Paramyxoviridae (parainfluenza virus type 3, mumps virus, measles virus), Reoviridae (reovirus type 3), Poxviridae (vaccinia virus), Herpesviridae (herpes simplex virus types 1 and 2), Togaviridae (Venezuelan equine encephalomyelitis virus, yellow fever virus, Japanese encephalitis virus), Bunyaviridae (Rift Valley fever virus, sandfly fever virus [strain Sicilian], Korean hemorrhagic fever virus), Arenaviridae (Pichinde virus), Picornaviridae (coxsackieviruses B1 and B4, echovirus type 6, encephalomyocarditis virus), Adenoviridae (adenovirus type 2), and Rhabdoviridae (vesicular stomatitis virus). The antiviral activity of selenazole was also cell line dependent, being greatest in HeLa, Vero-76, and Vero E6 cells. Selenazole was relatively nontoxic for Vero, Vero-76, Vero E6, and HeLa cells at concentrations of up to 1,000 ,g/ml. The relative plating efficiency at that concentration was over 90%. The effects of selenazole on viral replication were greatest when this agent was present at the time of viral infection. The removal of selenazole from the medium of infected cells did not reverse the antiviral effect against vaccinia virus, but there was a gradual resumption of viral replication in cells infected with parainfluenza type 3 or herpes simplex virus type 1 (strain KOS). However, the antiviral activity of ribavirin against the same viruses was reversible when the drig was removed.

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Binary combinations of the N-nucleoside ribavirin (1-3-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and the C-nucleoside analog selenazofurin (2-f8-D-ribofuranosylselenazole-4-carboxamide) or tiazofurin (2-(0-Dribofuranosylthiazole-4-carboxamide) were tested in vitro for activity against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, Rift Valley fever, Korean hemorrhagic fever, and Pichinde viruses. The 50% effective dose for each compound alone or in a series of combinations was determined with a plaque reduction assay. Combinations of ribavirin and selenazofurin were synergistic against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, and Pichinde viruses, with fractional inhibitory concentrations of 0.1, 0.2, 0.4, 0.4, respectively, but showed additive effects against Korean hemorrhagic fever and Rift Valley fever viruses. Combinations of ribavirin and tiazofurin were synergistic against yellow fever and Japanese encephalitis (fractional inhibitory concentrations, 0.41 and 0.48, respectively) but showed additive effects against Korean hemorrhagic fever virus. Combinations of selenazofurin and tiazofurin had additive effects against Japanese encephalitis, yellow fever, and Korean hemorrhagic fever viruses. The effect of combinations on cell toxicity was additive, both in monolayers of nondividing cells incubated under agar for the same period as the plaque assay and for rapidly dividing cells given short-term exposure (4 h), followed by determination of the proportion of surviving cells with a colony forming assay.In the search for more effective antiviral compounds, we have screened numerous N-glycosyl and C-glycosyl nucleosides against selected members of the toga-, bunya-, and arenavirus families. The carboxamide nucleoside ribavirin(1-p-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) (8) was one of the most effective broad-spectrum agents tested. Limited structural modification of ribavirin did not improve its antiviral activity significantly; however, several analogs have shown comparable antiviral activity. Included were the novel selenazole carboxamide nucleoside selenazofurin (2-13-D-ribofuranosyl-selenazole-4-carboxamide) (19), with a broad spectrum (13) and the closely related thiazole carboxamide nucleoside tiazofurin (2-1-D-ribofuranosly-thiazole-

Lipid-soluble spin labels were incorporated into human lymphocytes and mouse L-cells and the resulting electron spin resonance spectra were compared with spectra obtained from similarly labeled human erythrocytes. Spin labels were found in all subcellular fractions of the nucleated cells that contained membranes. Spinlabeled cells remained viable and capable of replicating in vitro. Electron spin resonance signals from spin-labeled nucleated cells underwent a time-and temperaturedependent decay that was reversed by bathing the cells in K3Fe(CN)6. The demonstration of a relative cell impermeability to ferricyanide, as measured by both colorimetric and radioisotopic label methods, indicated that only spin-labeled molecules in the surface membrane were reactivated when ferricyanide was added to spin-labeled cells after the electron spin resonance signal had decayed.Electron spin resonance (ESR) spectroscopy and spin-label techniques have been used recently to probe the structure of artificial membranes (1, 2) and membranes of erythrocytes (3), neurospora (4), viruses (5), lobster walking nerves (6), sarcoplasmic reticulum (7), and mycoplasma (8). However, information is not available regarding application of spinlabel techniques to studies with intact nucleated mammalian cells. A major problem in the application of ESR techniques to studies of surface membrane phenomena in intact cells is differentiating between labels attached to cell surface and those bound to internal membrane structures.This report describes a technique for differential detection of surface-membrane signals from spin-labeled, intact nucleated mammalian cells. The cellular distribution and spectral characteristics of various lipid-soluble spin labels incorporated in membranes of mouse L-cells and human lymphocytes are described. ESR spectra of these spin-labeled nucleated cells are compared with spectra of spin-labeled human erythrocytes. In addition, factors affecting signal stability and the effect of label incorporation on cell viability and replication are reported. MATERIALS AND METHODSSpin Labels. All spin labels were purchased from Syva Assoc., Palo Alto, Calif. The spin labels used were ( Fig. 1 in ME medium to 50% of the original volume of homogenate, the nuclear pellet was given three additional strokes in the Duall homogenizer and again centrifuged. Supernates were combined and centrifuged at 25,000 X g for 10 min to obtain a mitochondrial fraction (M), and at 48,000 X g for 60 min to obtain a microsomal fraction (P). The final supernate was the soluble fraction (S). N, M, and P fractions were concentrated by suspending them to 50% or 25% of the homogenate's original volume. Relative concentrations of label in each of the subcellular fractions were determined. 66