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Virus isolates resistant to 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(α-pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross-resistance evaluation indicated that the two drug resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400-resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross- resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400 Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (>20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds. Copyright 1995, 1999 Academic Press

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.

Transformation of rat embryo cells by murine sarcoma virus (MSV) was contingent upon synthesis of deoxyribonucleic acid (DNA) during the first 12 hr of infection. Inhibition of DNA synthesis by thymidine (20 mm) or cytosine arabinoside (0.1 mm) resulted in the protection of cells from transformation by MSV. Transient suppression of DNA synthesis prior to infection or after a 12-hr delay had little effect on subsequent transformation, emphasizing the critical time period in in which DNA synthesis was necessary for intracellular fixation of the viral genome. These results are similar to those previously described for Rous sarcoma virus. Development of transformed cells after viral fixation was shown to be influenced by cellular density. Under conditions which allowed fixation of virus in confluent cellular monolayers, less than 20% of these cells developed into transformed foci.

Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to 3H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain “defective” strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed.

A study of the metabolic requirements for the growth of psittacosis virus in L cells has been extended to the water-soluble vitamins. In a system in which a balanced salt solution was used to deplete the cells of their vitamin constituents, only thiamine was essential for psittacosis virus production. Extended depletion of cells with media deficient in specific vitamins demonstrated that pantothenate, niacin (niacinamide), pyridoxine (pyridoxal), and choline, in addition to thiamine, were essential for maximal growth of psittacosis virus. No requirement for biotin, inositol, folic acid, or riboflavin was demonstrated, although the possibility of incomplete vitamin depletion of the cells has not been eliminated. In most cases in which a specific vitamin requirement was shown the decreased yield of virus was correlated with a delay in the cytopathic effects produced in the cell cultures by psittacosis virus.

Mouse fibroblasts (L cells) fail to support the growth of psittacosis virus (6BC strain) if they are maintained on a medium containing only inorganic salts and glucose for 2 days prior to infection. Virus propagation can be stimulated by the addition of a synthetic medium containing amino acids, water-soluble vitamins, glutamine, glucose, and inorganic salts. By omitting single amino acids from the complete synthetic medium, tyrosine, threonine, methionine, isoleucine, phenylalanine, tryptophan, leucine, valine, and cysteine or cystine were found to be essential for stimulation, while lysine, arginine, histidine, hydroxyproline, proline, glutamic acid, aspartic acid, serine, alanine, and glycine were not essential. The cells on deficient media showed varying degrees of degenerative changes, but there was little correlation between ability to support psittacosis virus growth and morphologic condition of the cells. Glucose is also an essential component of the medium for viral growth, but the absence of glutamine had no effect on stimulation of virus propagation. L cell cultures maintained on media deficient in phenylalanine or tryptophan for 2 days before infection were also found to be incapable of supporting virus growth. The implications of this study in latent viral infections are discussed.

By maintaining L cells in a balanced salt solution of inorganic salts and glucose (BSS) for 2 days or more, they are rendered incapable of supporting the growth of psittacosis virus (6BC), though it infects such cells and is present intracellularly for as long as 3 days in a non-infectious phase. The addition of an enriched medium to such a culture of cells at any time up to 4 days after infection results in the appearance of infectious virus within these cells, which multiplies and is released from the cells, providing the entire period of exposure of such cells to the BSS does not exceed 6 days, following which the cells die. A latent infection with psittacosis virus in a non-infectious phase has been established in a pure line of cells which possess properties of malignancy.

Lactic dehydrogenase virus was grown in primary mouse embryo cells and labeled with 3H-uridine and 3H-amino acids. Concentrated and purified virus was banded by isopycnic centrifugation in sucrose gradients, and infectivity and radioactivity were found to correspond at a density of 1.17 g/cm3. The extracted viral RNA was resolved by electrophoresis in polyacrylamide-agarose mixed gels, and the mol wt was estimated to be 6.0 × 106.

Cells producing the Rauscher strain of murine leukemia virus (MLV) were exposed to 3H-uridine, and labeled virus was collected at hourly intervals. Ribonucleic acid (RNA) extracted from virions (vRNA) had a characteristic single peak when analyzed by electrophoresis in polyacrylamide-agarose composite gels. Exposure of vRNA to dimethyl sulfoxide, urea, formaldehyde, or heat altered the mobility to a faster moving form (vRNA'). This vRNA' sedimented more slowly than native vRNA in sucrose gradients. Incubation of labeled virions at 37 C resulted in fragmentation of viral RNA which was detectable only after denaturation. Also, large differences in the temperature required for the change from vRNA to vRNA' were seen with alterations in NaCl concentration. These experiments demonstrate that the vRNA of MLV is held in a specific conformation by hydrogen bonds distributed over a large part of the molecule. The possibility that an undefined factor is associated with viral RNA is discussed.

The ribonucleic acid (RNA) of murine leukemia virus (MLV) Rauscher strain was observed by the aid of electron microscopy with the use of the protein monolayer technique. RNA was observed directly after release from virus particles or after isolation by sedimentation in sucrose density gradients. Molecules were found in an extended linear form. Many of the RNA filaments released by detergent treatment contained curled regions, suggesting the linear filaments were originally coiled within the virus particle. The relationship of the curled areas to the containment of the RNA within the virus particle is discussed, and a mechanism for the inclusion of RNA in the budding virion is proposed. Treatment of the extended MLV-RNA with dimethyl sulfoxide resulted in the collapse of the molecule forming a tangled complex. Treatment with urea or heating at 50 C in 3 mmNaCl also produced this effect. Also under the conditions in which MLV-RNA was linear, RNA from Rous sarcoma virus also was linear, but Newcastle disease virus RNA and ribosomal RNA of rat liver had collapsed structures. The results indicated that the RNA of MLV, and perhaps other RNA-containing tumor viruses, has a specific unique conformation dependent upon hydrogen bonds.