Cycloheximide induction of xenotropic type C virus from synchronized mouse cells: metabolic requirements for virus activation.

The information for type C RNA viruses is genetically transmitted within the cellular DNA of the normal mouse cell. These viruses can be induced after exposure of cells to two classes of chemicals, inhibitors of protein synthesis and halogenated pyrimidines. The metabolic requirements for activation of one endogenous virus of BALB/c mouse cells by representatives of each class of drugs were studies. Cycloheximide and iododeoxyuridine each induce virus efficiently from cultures in exponential growth but are inactive on cells in stationary phase. However, cells are maximally sensitive to the actions of each drug at different times within the cell cycle. Further, virus induction in response to each is differentially inhibited under conditions of simultaneous cell exposure to inhibitors of DNA or RNA synthesis. The results provide support for the concept that inhibitors of protein synthesis and halogenated pyrimidines act by different mechanisms to induce type C virus release.     

Structure of bovine papillomavirus type 1 DNA in a transformed mouse cell line

Linearized bovine papillomavirus type 1 (BPV-1) DNA was introduced into mouse C127 cells, where it recircularized and replicated as an intact monomeric, extrachromosomal circular form in the resulting transformants. These cells contained a mixture of complex high molecular weight forms that were converted to a linear form of approximately BPV-1 size upon digestion with an enzyme that cuts once within the BPV-1 genome. Further analysis of one of these cell lines revealed that these high molecular weight forms consisted of two components. One was detected on agarose gels as a diffuse smear of slow-migrating material representing linear forms that were tightly associated with host chromosomes, probably by integration. The second component was composed of discrete-sized oligomeric open and supercoiled extrachromosomal circular forms of up to approximately 48 X 10(3) base-pairs (6 tandemly linked BPV-1 genomes) in size. No catenated (interlocked) forms could be detected.     

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells expressed an 85,000-dalton protein that was precipitated by antisera directed against feline leukemia virus p12, p15, and p30 proteins. No feline oncornavirus-associated cell membrane antigen reactivity was detected on the surfaces of the transformed murine cells by indirect membrane immunofluorescence techniques. The 85,000-dalton feline sarcoma virus-specific protein was also found in feline cells transformed by transfection. However, these cells also contained env gene products. The results of this study demonstrate that the feline sarcoma virus genome is sufficient to transform murine cells and that expression of the 85,000-dalton gag-x protein is associated with transformation of both murine and feline cells transformed by transfection.     

Isolation from BALB/c mouse cells of a structural polypeptide of a third endogenous type C virus

A 12,000 molecular weight type C viral polypeptide, p12, has been isolated from BALB/c mouse cells. This polypeptide is shown to be immunologically distinct from the p12 antigens of two previously described endogenous viruses of BALB/c cells. It is, however, indistinguishable from a viral antigen expressed in NIH Swiss mouse cells and present in type C viruses isolated from NIH Swiss mice. The expression of endogenous viruses containing each of the three distinguishable p12 antigens is shown to be differentially affected by two classes of chemical inducers, halogenated pyrimidines and inhibitors of protein synthesis. The present findings thus provide evidence for the existence of genetic information of three distinguishable endogenous viruses within cells of the BALB/c strain.     

Friend strain of spleen focus-forming virus: a recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus.

The genome of the Friend strain of the spleen focus-forming virus (SFFV) has been analyzed by molecular hybridization. SFFV is composed of genetic sequences homologous to Friend type C helper virus (F-MuLV) and SFFV-specific sequences not present in F-MuLV. These SFFV-specific sequences are present in both the Friend and Rauscher strains of murine erythroleukemia virus. The SFFV-specific sequences are partially homologous to three separate strains of mouse xenotropic virus but not to several cloned mouse ecotropic viruses. Thus, the Friend strain of SFFV appears to be a recombinant between a portion of the F-MuLV genome and RNA sequences that are highly related to murine xenotropic viruses. The implications of the acquisition of the xenotropic virus-related sequences are discussed in relation to the leukemogenicity of SFFV, and a model for the pathogenicity of other murine leukemia-inducing viruses is proposed.     

Biological properties of a type C virus isolated from a human X mouse hybrid cell line.

The biological properties of the HMV-1 virus, spontaneously released from a human X C57BL/6 mouse hybrid cell line, were similar to those of RadLV, the prototype B-tropic virus of C57BL/6 mice. Both viruses replicated on B-type mouse cells and in the wild mouse cell line SC-1. The plaque-forming abilities of the two viruses were relatively low, but gradually increased after passage in new host cells. Both viruses were neutralized by AKR antisera but not by FMR antisera. HMV-1 virus could rescue the defective sarcoma genome from S+H- mouse cells. The pseudotype sarcoma virus so produced was deficient in "helper virus" activity. Newborn mice inoculated with HMV-1 virus remained tumor-free over a 1-yr observation period.     

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.     

Characterization of infectious hepatitis C virus from liver-derived cell lines

The efficient production of infectious HCV from the JFH-1 strain is restricted to the Huh7 cell line and its derivatives. However, the factors involved in this restriction are unknown. In this study, we examined the production of infectious HCV from other liver-derived cell lines, and characterized the produced viruses. Clones of the Huh7, HepG2, and IMY-N9, harboring the JFH-1 full-genomic replicon, were obtained. The supernatant of each cell clone exhibited infectivity for naïve Huh7. Each infectious supernatant was then characterized by sucrose density gradient. For all of the cell lines, the main peak of the HCV-core protein and RNA exhibited at approximately 1.15 g/mL of buoyant density. However, the supernatant from the IMY-N9 differed from that of Huh7 in the ratio of core:RNA at 1.15 g/mL and significant peaks were also observed at lower density. The virus particles produced from the different cell lines may have different characteristics.     

Recovery of biologically active spleen focus-forming virus from molecularly cloned spleen focus-forming virus-pBR322 circular DNA by cotransfection with infectious type C retroviral DNA.

The genome of the Lilly-Steeves strain of spleen focus-forming virus (SFFV) was molecularly cloned in the plasmid vector pBR322. Infectious SFFV could be recovered by releasing the SFFV DNA from the vector, transfecting the released DNA onto NIH 3T3 cells, and rescuing the SFFV either by superinfection with helper virus or by cotransfection with molecularly cloned infectious helper viral DNA. By using transfections with SFFV DNA still attached to the plasmid vector, infectious SFFV activity could also be recovered with either method of rescue. Studies performed with these latter types of transfections indicated that only a portion of the SFFV genome was required for biological activity. Since gp52, a marker protein for SFFV, could be detected in all cultures from which adequate titers of biologically active SFFV were recovered, the results are consistent with the hypothesis that gp52 is necessary for SFFV-induced erythroblastosis and polycythemia.     

Introduction of the herpes simplex virus thymidine kinase gene into mouse cells using virus DNA or transformed cell DNA.

Cells lacking the enzyme thymidine kinase (LMTK- cells) have been transformed to a kinase-positive phenotype using sheared herpes simplex virus (HSV) DNA, and the enzyme found in these transformed cells is HSV-specific. One of the cell lines is able to complement the functional defect found in two temperature-sensitive mutants of HSV 1, and reversion of the cells to a thymidine kinase-negative phenotype results in the loss of this capability. The HSV thymidine kinase gene can also be introduced into LMTK- cells using DNA extracted from transformed cells, and the high efficiency of this procedure suggests that the state of the virus DNA in transformed cells is different from that of DNA in virus particles.     

Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.     

Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein.

A search for variant endogenous cat viruses led to a novel isolate. Although the major envelope glycoprotein of this virus was similar in size to that of an RD-114-like virus that was coisolated, it was unrelated to RD-114 or feline leukemia virus by immunological and biological criteria. This degree of dissimilarity suggests a different evolutionary progenitor from that for the RD-114 and feline leukemia virus viral envelopes. The novel virus did, however, code for gag gene polypeptides which are closely related to RD-114 virus. Neither the novel isolate nor the RD-114-like coisolate induced foci in S+L- cat cells which restrict focus induction by RD-114 virus. This suggests that the two viruses share a common genomic target of restriction which resides outside of the env region.