Transformation of chondroblasts by Rous sarcoma virus and synthesis of the sulfated proteoglycan matrix.

The presence of the extracellular matrix synthesized by chondroblasts provides a barrier to virus penetration. Chondroblasts can be infected and transformed following treatment with proteolytic enzymes. Using a temperature-sensitive transformation mutant of Rous sarcoma virus and rearing the cells at permissive temperature, we demonstrate that transformed chondroblasts stop synthesizing their cell-unique sulfated proteoglycan. If such transformed chondroblasts are shifted to nonpermissive temperature, the cells reinitiate the synthesis of their cell-unique sulfated proteoglycan.     

Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of Rous sarcoma virus.

Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.     

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.     

Cells transformed by temperature-sensitive mutants of avian sarcoma virus cause tumors in vivo at permissive and nonpermissive temperatures.

Chick embryo (CE) fibroblasts and normal rat kidney (NRK) cells transformed by temperature-sensitive (ts) mutants of avian sarcoma virus (NY68, LA23, LA24, LA25, LA29, LA31, GI201, GI202, GI251, GI253 induce tumors on the chorioallantoic membrane (CAM) of chick eggs at temperatures that correspond to the permissive and nonpermissive temperatures used to induce conditional expression of the "transformed" phenotype in these cells when cultured in vitro. Chick embryo cells infected with transformation-defective mutants of ASV (td101, td108) or RAV-50 were nontumorigenic under the same conditions, as were nontransformed CE and NRK cells. This indicates that the CAM is not an unusually susceptible substrate for cell growth and that the ability of tsASV-transformed cells to form tumors at nonpermissive temperatures reflects their true tumorigenicity. In contrast, a ts mutant chemically transformed rat liver cell line, ts-223, only formed tumors on the CAM under permissive conditions. The wild-type parent cells (W-8) of this mutant produced tumors at both permissive and nonpermissive temperatures. Direct implantation of microprobe thermometers into tumors caused by ts-ASV-transformed cells at nonpermissive temperatures confirmed that tumor formation occurred in a stable temperature environment and was not due to temperature fluctuations which might have created semi-permissive conditions for tumor growth. Cells isolated from tumors formed at nonpermissive temperatures and recultured in vitro displayed temperature-dependent hexose transport and colony formation in agar similar to the orginal parent cell inoculum. Similarly, virus recovered from tumors at nonpermissive temperatures retained the ts mutation.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2.

BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.     

Action of temperature-sensitive mutants of myeloproliferative sarcoma virus suggests that fibroblast-transforming and hematopoietic transforming viral properties are related

The myeloproliferative sarcoma virus is molecularly related to the Moloney sarcoma virus (Pragnell et al., J. Virol. 38:952-957, 1981), but causes both fibroblast transformation in vitro and leukemic changes--including spleen focus formation--in adult mice. The fibroblast transforming properties of myeloproliferative sarcoma virus were used to select viral temperature-sensitive mutants at 39.5 degrees C, the nonpermissive temperature. These mutants are temperature sensitive in the maintenance of the transformed state. This was also shown by cytoskeletal changes of the infected cells at permissive and nonpermissive temperatures. Viruses released from cells maintained at both the permissive and nonpermissive temperature are temperature sensitive in fibroblast transformation functions. All temperature-sensitive mutants show only a low reversion rate to wild-type transforming function. The myeloproliferative sarcoma virus temperature-sensitive mutants are inefficient in causing leukemic transformation (spleen enlargement, focus formation) in mice at the normal temperature. A method to maintain a low body temperature (33 to 34 degrees C) in mice is described. One temperature-sensitive mutant was checked at low body temperature and did not induce leukemia. These data thus indicate that the same or related viral functions are responsible for hematopoietic and fibroblast transformation.     

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.     

A genetic test for expression of a functional herpes simplex virus DNA-binding protein from a transfected plasmid.

The major DNA-binding protein, ICP8, encoded by herpes simplex virus is localized to the infected cell nucleus where it plays a role in viral DNA replication and control of viral gene expression. To identify the parts of the ICP8 protein that are important for its localization and functions, we have developed a system to test the ability of recombinant plasmids to express functional ICP8. A recombinant plasmid containing the wild-type ICP8 gene was transfected into cells. The cells were later infected with a temperature-sensitive ICP8 mutant virus at the nonpermissive temperature. Sufficient wild-type ICP8 was expressed from the transfected plasmid to complement the replication of the mutant virus. This provides a genetic system to test the properties of ICP8 expressed from mutagenized plasmids without the establishment of a stable cell line or the reintroduction of the ICP8 gene into the herpes simplex virus genome.     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.     

Distinguishable transformation-defective phenotypes among temperature-sensitive mutants of Rous sarcoma virus.

Eight transformation-defective, temperature-sensitive (ts) mutants of the Prague strain of Rous sarcoma virus, subgroup A, have been isolated after mutagenesis with 5-bromodeoxyuridine followed by selection on the basis of focus tests. Five of these mutants, ts GI201, GI202, GI203, GI204, and GI205, exhibit properties like most previously reported isolates in that they show a temperature-sensitive response to each of a variety of transformation-specific parameters tested. Interestingly, GI201, in addition to the temperature-sensitive defect, carries a lesion that was observed as a nonconditional loss of expression of plasminogen activator protease. Three mutants, ts GI251, GI252, and GI253 have been disignated partial transformation-defective (PTD) mutants since they behave as ts mutants according to some tests for transformation and as wild type according to others. These three mutants fail to form foci at the nonpermissive temperature (41 degrees C) and art nontumorigenic in 3-week-old chickens (body temperature, 42 degrees C). The agglutinability by concanavalin A of cells infected with these mutants shows a definite temperature sensitivity, as do the rate of 2-deoxyglucose uptake and the disappearance of the 250, 000-dalton normal cell glycoprotein (large, external, transformation sensitive [LETS]). Although the PTD mutant-infected cells, unlike cells infected with other transformation mutants, exhibit a cell-bound plasminogen activator protease at the nonpermissive temperature, this activator is not detectable as a free protease in the medium, as it is with wild-type, virus-infected cells. The PTD mutants behave like the wild-type parent in their ability to induce transformed growth properties in the infected cells, i.e., growth beyond normal cell saturation density with or without serum-supplemented medium and growth leading to colony formation in soft-agar- or methyl cellulose-containing suspension media.     

Mutant of Vesicular Stomatitis Virus Which Allows Deoxyribonucleic Acid Synthesis and Division in Cells Synthesizing Viral Ribonucleic Acid

Some temperature-sensitive mutants of vesicular stomatitis virus were tested for their ability to block the initiation of deoxyribonucleic acid (DNA) synthesis and division in serum-stimulated hamster embryo fibroblasts at the nonpermissive temperature. Although the parental strain blocked these processes, one particular mutant allowed essentially normal DNA synthesis and division. By autoradiography, it was shown that individual cells infected with this mutant could synthesize viral ribonucleic acid and at the same time initiate DNA synthesis and divide. Cells infected with such conditional defective mutants appear to be suitable for studies on the effects of persistent viral infections on molecular and cellular functions in proliferating cell populations.     

Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein

Maturation of the vesicular stomatitis virus (VSV) glycoprotein (G) to the cell surface is blocked at the nonpermissive temperature in cells infected with temperature-sensitive mutants in the structural gene encoding for G. We show here that these mutants fall into two discrete classes with respect to the stage of post-translational processing at which the block occurs. In all cases the mutant glycoproteins are inserted normally into the endoplasmic reticulum membrane, receive the two-high-mannose oligosaccharides, and apparently lose the NH2-terminal signal sequence of 16 amino acids. In cells infected with one class of mutants, no further processing of the glycoprotein occurs, and we conclude that the mutant protein is blocked at a pre-Golgi stage. In cells infected with ts L511(V), however, addition of the terminal sugars galactose and sialic acid occurs normally. Thus the maturation of G proceeds through several Golgi functions but is blocked before its appearance on the cell surface. The oligosaccharide chain of ts L511(V) G, accumulated at either the permissive (where surface maturation occurs) or the nonpermissive temperature, lacks one saccharide residue, probably fucose. In addition, no fatty acid residues are added to the ts L511(V) G protein at the nonpermissive temperature, although addition does occur under permissive conditions.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.