Efficient recovery and regeneration of integrated retroviruses.

We report a rapid and efficient PCR-based rescue procedure for integrated recombinant retroviruses. Full-length proviral DNA is amplified by long-range PCR using a pair of primers derived from the long terminal repeats (LTR), and virus is regenerated by transfecting retrovirus-packaging cells with the PCR-derived provirus. The viral yield from the PCR product is similar to that from the retroviral plasmid vector, and the representation of different inserts is accurately maintained in the recovered retroviral population. This procedure is suitable for expression cloning from retroviral libraries and should be applicable to the analysis of natural retrovirus populations.

An overview of retrovirus replication and classification.

This introductory chapter has presented an overview of how retroviruses replicate and how they are classified within the family Retroviridae. The genomic structure of retroviruses, so reminiscent of bacterial transposons and other similar genetic elements, and reverse transcriptase, which leads to the reverse flow of genetic information from RNA to DNA, are responsible for many of the properties of these viruses which make them both fascinating and important as causes of cancer and other diseases. The requirement for integration shared by most retroviruses leads directly to most of the phenomena resulting from their interaction with target cells. Certainly latency, at the level of the organism, is one such property relevant to how we think of vaccines and therapeutic reagents. The ability of retroviruses to acquire oncogenes from cellular DNA has greatly facilitated our understanding of the genetics of neoplasia. Additionally, the use of retroviral vectors to introduce new genes into genetically defective animals is a consequence of the genetic organization of retroviruses. Classification of viruses at the species level is difficult for several reasons. In particular, viruses do not sexually reproduce in any conventional sense, and it is difficult to identify a population of virions which make up a genetically distinct pool. Thus, the definition of individual species is often controversial and is not necessarily aided by the criteria used to define larger phylogenetic groups. In the latter case, retroviruses have distinctive morphological and biochemical features which allow their classification at the family, subfamily, genus, and subgenus levels. Additional classification occurs by accounting for factors such as host range, cross neutralization, ability to compete in interspecies radioimmunoassays, and genetic homology detected by hybridization under conditions of relaxed stringency. Direct comparison of nucleotide sequences offers the hope that mathematical criteria will be developed that can define the level of differences characteristic of individual species, genuses, and subfamilies.

Biologic and molecular characterization of two newly isolated ras-containing murine leukemia viruses.

A murine sarcoma virus (MSV) was recovered from an (NFS X NS.C58v-1) F1 mouse which developed splenic sarcoma and erythroleukemia 6 months after inoculation with a mink cell focus-inducing murine leukemia virus (MuLV) isolated from an NFS mouse infected with a wild mouse ecotropic MuLV. The MSV, designated NS.C58 MSV-1, induced foci of transformation in mouse and rat fibroblasts, and inoculation of mice of various strains 2 weeks of age or younger resulted in erythroleukemia and sarcomatous lesions in spleen, lymph node, and brain. The MSV provirus was molecularly cloned from a genomic library prepared from transformed non-producer rat cells. The 8.8-kilobase proviral DNA contained a 1.0-kilobase p21 ras coding segment which replaced most of the gp70-encoding portion of an MuLV, most likely the endogenous C58v-1 ecotropic virus. The ras oncogene is closely related to v-Ha-ras by hybridization, expression of p21 protein, and nucleotide sequence. It is nearly identical in sequence to v-bas, the only previously described transduced, activated mouse c-ras. At position 12 in the p21 coding region, arginine is substituted for the naturally occurring glycine present in c-ras. A second MSV isolate is described which is similar to NS.C58 MSV-1 except for a 100- to 200-base-pair deletion in the noncoding region of the ras-containing insert.

Isolation from the spleen of myeloproliferative sarcoma virus-infected mice of a myelomonocytic tumor cell line secreting hematopoietic colony stimulating factors.

Clonogenic tumor cells were detected in the spleen of DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). These cells could be isolated because of their ability to proliferate on the greater omentum of sublethally irradiated isogenic recipient mice. We have established a permanent suspension myelomonocytic cell line in vitro (TE8), from a tumor obtained after subcutaneous transplantation of the omental tumor masses. The TE8 cells are clonogenic in semi-solid medium containing agarose. The colonies thus obtained gave rise to myelomonocytic cell lines growing in suspension in liquid medium. One of those cloned cell lines, C1(1011) was studied in details. It is exclusively composed of initial donor cells, it is tumorigenic in vivo, clonogenic in vitro and produces MPSV. It also secretes a colony stimulating activity (CSA), and an activity termed mixed colony promoting activity (MPA), which enables pluripotent hematopoietic stem cells to proliferate and differentiate. It differentiates through the granulomacrophage cell line independently of exogenous factors other than those which may be present in the serum.

Xenotropic C-type viruses and autoimmune disease.

New Zealand Black (NZB) mice develop a disease complex which resembles autoimmunity in man. Cells from both NZB embryos and adults spontaneously produce an infectious C-type virus which is xenotropic (X-tropic)--capable of being passed only to cells foreign to the host species. It differs from other endogenous murine C-type viruses which are ecotropic, i.e. infectious for cells of their home species. Xenotropic viruses are detected in embryos and adult tissues of other strains of mice but at less frequency and titer than in NZB mice. Moreover, natural anti-X-tropic virus neutralizing activity, which is present in all mouse sera, is found in significantly higher titers in NZB mouse sera. A working hypothesis is that the X-tropic virus is an important agent for normal development and differentiation, but in NZB mice its increased expression results in autoimmune disease. Furthermore, its interaction with endogenous ecotropic virus leads to phenotypic mixing with possible enhancement of the immunologic disorders.

[Isolation and characterization of retroviruses expressed in murine osteosarcomas induced by 90Sr]. / Isolement et caractérisation de rétrovirus exprimés dans les ostéosarcomes murins induits par le 90Sr.

The induction of osteosarcomas with 90Sr in CF1 mice is associated with the expression of ecotropic type-C RNA viruses devoid of sarcomatogenic activity. In contrast, the FBR murine osteosarcoma virus complex, isolated from a 90 Sr-induced osteosarcoma of a X/Gf mouse [M. Finkel et al. (1)], causes the rapid appearance of osteosarcomas in newborn mice and transforms fibroblasts in vitro. The transforming capacity of FBR murine sarcoma virus has been associated as an oncogene homologous to v-fos.

Isolation and characterization of a Mo-MuSV-transformed TB cell line that produces noninfectious MuSV particles with uncleaved gag protein which is processed in the presence of Mo-MuLV.

A cell line, TBSV7, that produces noninfectious murine sarcoma virus (MuSV) in the absence of helper MuLV was isolated from TB cells infected with the supernatant of MuSV349 cells. These noninfectious MuSV particles with "immature" C-type virus morphology contain a 2.2 X 10(6)-Da genomic RNA and an uncleaved 62,000-Da gag precursor protein (Pr62). Neither viral envelope proteins (gp70, p15E, p12E) nor reverse transcriptase were detected in these virus particles. Pr62 was found to be phosphorylated in vivo and it could be phosphorylated in vitro with [gamma-32P]ATP, indicating that protein kinase was packaged in these noninfectious virions. In vitro processing of Pr62 to smaller molecular weight proteins could be achieved by the addition of Mo-MuLV and Nonidet P-40. The initial cleavage products were proteins with molecular weights of 38K (Pr38) and 27K (Pr27). Under optimum conditions Pr38 was cleaved to p30 and a protein band migrating with MuLV-p10, while Pr27 was cleaved to a 17,000-Da protein that migrated slower than MuLV-p15 and a protein band migrating with MuLV-p12. Pulse-chase experiments performed on TBSV7 cells superinfected with Mo-MuLV indicated that intracellular processing of Pr62 was much slower than that of Pr65. Cleavage protein products of Pr62 similar in size to the in vitro protein products were also detected in TBSV7 cells superinfected with MuLV.

New mammalian transforming retrovirus: demonstration of a polyprotein gene product.

A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.

Growth of murine sarcoma and murine xenotropic leukemia viruses in Japanese quail: induction of tumors and development of continuous tumor cell lines.

The BALB/c murine endogenous xenotropic leukemia virus pseudotype of m1 Moloney sarcoma virus [m1MSV(B-MuX)] was used to productively transform diploid Japanese quail embryo cells. The majority of newly hatched quail inoculated with m1MSV(B-MuX)-transformed quail embryo fibroblast cells rapidly developed tumors which were predominantly locally invasive fibrosarcomas, but metastases were observed in two birds. In two tumor-bearing quail, lymphosarcomas were observed in conjunction with fibrosarcomas. Quail inoculated with m1MSV(B-MuX) virus or quail embryo fibroblast cells infected with helper leukemia virus did not develop tumors. A cell culture derived from one quail tumor was shown to be oncogenic in newly hatched quail. Viruses produced by m1MSV(B-MuX)-infected quail cells or quail tumors had envelope properties of BALB-derived murine xenotropic leukemia virus as measured by host range, interference, and neutralization. Virus structural antigens, proteins with molecular weights of 30,000 and 70,000, were detected in tumors and tumor-derived cell lines by immunofluorescence and gel diffusion. Sera from tumored quail had high titers of type-specific BALB-derived murine xenotropic leukemia virus antibodies as determined by neutralization and immunoprecipitation. Antibodies to the putative m1 Moloney sarcoma virus mos gene product were not detected in sera from tumored or regressor quail.

Origin and biological properties of a new BALB/c mouse sarcoma virus.

A focus-forming virus previously isolated from a BALB/c mouse hemangiosarcoma has been shown to be replication defective. Analysis of individual BALB/c mouse sarcoma virus (BALB-MSV) nonproducer transformants for expression of helper virus-coded proteins revealed genetically stable variants that expressed two, three, or all four gag gene products in the absence of detectable helper viral env gene expression. The type-specific antigenic determinants of helper viral proteins encoded by the BALB-MSV genome and by the B-tropic virus isolated from the BALB-MSV stock were demonstrated to be indistinguishable from those of BALB:virus-1, a known endogenous virus of BALB/c cells. These findings imply that a BALB/c endogenous virus was involved in the generation of BALB-MSV. By the same immunological approach, the presence of at least a portion of the Moloney-MuLV gag gene has been identified in two other transforming viruses--Moloney-MSV and Abelson lymphosarcoma virus--previously isolated from the BALB/c strain. The tissue culture properties of cells transformed by these defective viruses were also shown to be distinguishable. These findings indicate that transforming virus isolates of the same inbred strain differ in their transforming activities as well as in the helper viral sequences stably associated with their genomes.

Rescue of a transforming virus from a spontaneous nonproducing osteosarcoma in BALB/c mice.

Cells from spontaneous osteosarcoma V793 that originated in a 19-month-old female BALB/c mouse were cultured. They did not produce a C-type oncovirus as determined by extracellular reverse transcriptase assay and cytoplasmic immunofluorescence. After cocultivation with Balb/3T3 cells chronically infected with a murine leukemia virus (MuLV), a focus-forming principle that transformed 3T3 cells, secondary BALB/c mouse embryo and WAG/Rij rat embryo fibroblasts were rescued. The transformation could be inhibited by antiserum to MuLV.

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus.

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A,1 rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A280 units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.

Transformation of rat liver epithelial cells by Kirsten murine sarcoma virus.

We studied the transformation of epithelial, diploid cell lines (RL-33 and RL-34) derived from W rat liver by the Kirsten murine sarcoma virus. On days 4-5 after virus infection, the epithelial cells began to pile up focally, forming small projections and releasing round cells from the foci. The epithelial cells grew in chains or as islets and grew in suspension above the cells attached to the bottom of the flasks when the cultures reached the confluent stage. The virus titration pattern was "one-hit." Three classes of transformed cells were isolated with respect to virus release and antigen expression: 1) virus producer, 2) non-producer, and 3) sarcoma-positive, leukemia-negative cells. When transplanted sc into newborn rats, the transformed cells produced sarcomas. The transformed cells formed within 1-3 days larger aggregates than those of their normal counterpart cells when suspended in liquid growth medium above an agar base. Aggregate properties (size, viability, and proliferation) of transformed cells correlated with growth in soft agar and tumorigenicity. RNA-dependent DNA polymerase and type C virus particles were readily induced in the normal rat liver epithelial cells after exposure to 5-iodo-2'-deoxyuridine.

An improved assay for feline leukaemia virus pseudotypes of murine sarcoma virus.

An assay is described for feline leukaemia virus pseudotypes of murine sarcoma virus which increased the virus titre by about 100-fold over conventional assays. The titre is independent of dilution and no secondary focus formation occurs. The assay may be used to study virus neutralization and to detect and type feline leukaemia virus in feline embryo cells by interference.

[Isolation of murine sarcoma virus not associated with XC plague forming leukemia virus]. / Isolement d'un virus du sarcome murin non associé á un virus de la leucémie murine formateur de plages dans le test XC

The N-type MLg cells transformed by B-tropic MuSV (WN1802B) produced defective MuSV without detectable among of MuLV; the focus formation by the culture fluid required the co-infection of exogenous MuLV. After one passage of the virus in MLg cells, we obtained a virus preparation which was capable of inducing foci without the exogenous MuLV, but which remained negative in the XC test.

Sarcoma-negative leukemia-positive transformed cell culture established from a murine sarcoma virus-induced rat bone tumor.

Inoculation of the Soehner-Dmochowski isolate of the Moloney strain of murine sarcoma virus (MSV), designated MSV-SD, consistently leads to the development of bone tumors in the susceptible New Zealand black (NB) rats. Two separate cell cultures have been established from 2 individual MSV-SD-induced NB rat bone tumors. Cells of 1 bone tumor culture, designated RBT-E, are in early in vitro passages. These cells form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at a greatly enhanced rate, 5 times that of normal nontransformed rat embryo cells. Cells of the RBT-E culture release both MSV and murine leukemia virus (MuLV) and therefore contain sarcoma-positive leukemia-positive transformed cells. The other rat bone tumor culture, designated RBT-L, produced MSV at early passages. RBT-L culture has been passaged over 130 times in vitro. Cells of the RBT-L culture form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at an enhanced rate (3 times that of rat embryo cells), indicating the presence of transformed cells within the RBT-L culture. However, cells of the RBT-L culture at late passages (Passage 130 or more) produce only MuLV and no detectable MSV activity (as shown by the lack of tumor-inducing activity and the lack of focus-forming activities by direct assay or by infectious center assay). Attempts to rescue MSV activity from RBT-L cells by cocultivation with MuLV-producing mouse cells were not successful. The MuLV found in the RBT-L cells, however, is a competent helper virus capable of rescuing the MSV genome from MSV-SD-induced hamster bone tumor cells. All the available evidence supports the notion that late passages of the RBT-L culture contain transformed cells that do not produce conventionally detectable MSV. These cells are referred to as sarcoma-negative leukemia-positive cells. The sarcoma-negative leukemia-positive cells represent a different kind of MSV-induced transformed cells and provide a unique system for studies in search of MSV markers such as MSV-specific antigens and MSV-specific nucleotide sequences.