Murine xenotropic type C viruses. V. Biologic and structural differences among three cloned retroviruses isolated from kidney cells from one NZB mouse

Three xenotropic retroviruses have been biologically cloned from cells cultured from the kidney of a 3-month-old NZB female mouse. They were obtained by first cocultivating the kidney cells for several weeks with mink, dog, and human cells and then cloning them by endpoint dilution. The cloned viruses differ in their infectivity and replicative ability in a variety of heterologous cell lines. The mink cell line-derived virus (X-NZB/K-Mlc) reaches titers in culture of over 10(8) infectious viruses/ml, and is produced in high titer within 24 hr after infection of mink lung cells. The human and dog cell-derived NZB viruses (X-NZB/K-Huc and X-NZB/K-Dgc) grow to lower titers and are similar in many respects. They differ in their relative ability to replicate in dog and human cells and to transform mink S+L- cells. Peptide mapping studies indicate that the X-NZB/K-Mlc virus has a unique p15(E) protein which distinguishes it from the other two cloned NZB viruses. These results lend further support to the observation that several types of xenotropic virus are present in a mouse strain and that more than one virus can be expressed by one organ of a particular mouse.     

A transfection assay for transformation by feline sarcoma virus proviral DNA

We report that fibroblast lines derived from mouse (NIH 3T3) and mink (CCL-64) can be transformed via transfection with the total cellular DNA extracted from cells transformed with feline sarcoma virus. Replication of helper virus is not required in the recipient cell. Transformation of recipient cells is observed when the donor DNA is extracted from either virus producer or nonproducer transformed cell lines. A transforming virus can be rescued from nonproducer transformed recipients upon superinfection with a replication competent helper virus.     

Mink cell line Mv 1 Lu (CCL 64). Focus formation and the generation of "nonproducer" transformed cell lines with murine and feline sarcoma viruses

Because of its flat regular growth in monolayer culture and its broad host range as a permissive cell for replication of a variety of mammalian type C viruses, the MvlLu mink cell line (CCL 64) has been found to be useful for focus forming assays of murine and feline sarcoma viruses, the generation of nonproducer murine and feline sarcoma virus-transformed cell lines, and the construction of sarcoma virus pseudotypes of mammalian type C viruses.     

Characterization of an amphotropic murine C-type virus that is NB-tropic.

Cocultivation of SIPC-2 myeloma cells with clone A₃₁, BALB/3T3 cells results in the continuous production of a C-type virus by the A₃₁ cells that has been designated S/A-1. S/A-1 virus possessed the essential properties of a murine retrovirus, including a buoyant density of 1.16 g/cm³, C-type morphology, high molecular weight (70 S) RNA, and an RNA-dependent DNA polymerase. After cloning by three endpoint dilution passages in A₃₁, and SIRC cells, the virus was shown to possess ecotropic as well as xenotropic activity. For example, the S/A-1 virus producibility infected mouse cells, including BALB/3T3, NIH/3T3, SC-1, 3T3 FL, and F₁ cells as well as non-mouse cells, including normal rat kidney, rabbit corneal (SIRC), Chinese hamster peritoneal, mink lung, Pekin duck embryo, human rhabdomyosarcoma, and guinea pig embryo cells. The S/A-1 virus infected A₃₁, cells do not induce XC-syncytia and the virus does not induce foci in mink lung cells. Thus, the data suggest that it is an amphotropic virus whose ecotropism is different (i.e., NB-tropic) from the previously reported amphotropic viruses (i.e., N-tropic). In addition, S/A-1 virus is different from the mink cell focus-inducing (MCF) viruses (J. W. Hartley, N. K. Wolford, L. J. Old, and W. P. Rowe 1977, Proc. Nat. Acad. Sci. USA74, 789–792) in terms of its lack of focus-inducing activity on mink lung cells, B-tropism, and ability to replicate in human rhabdomyosarcoma and F₁ mouse cells. The structural proteins of cloned S/A-1 virus were compared to those of 1504-A virus, a previously described amphotropic virus isolated from feral mice (J. W. Hartley and W. P. Rowe 1976, J. Virol.19 19–26; S. Rasheed, M. B. Gardner, and E. Chan 1976, J. Virol.19, 13–18), by electrophoresis on a linear 7–20% acrylamide slab gel and staining with Coomassie blue. S/A-1 differed from 1504-A in terms of (a) the mobility of a protein with a molecular weight of approximately 12,000 daltons (pl5) and (b) the fact that it has a “double” p30.     

Homologies among the nucleotide sequences of the genomes of C-type viruses

DNA was synthesized with detergent-disrupted virions of several C-type viruses and used to measure the extent of homology among the genomes of these viruses by molecular hybridization. The DNA was reacted with viral RNA under conditions which permit saturation of most if not all complementary nucleotide sequences in the RNA. This technique provides a quantitative estimate of the extent of homology among viral RNAs and is superior to current procedures that measure the fraction of DNA hybridized to an excess of viral RNA. The genomes of RD-114 and Crandell virus are at least 85% related, whereas there is no detectable homology among the genomes of RD-114 virus, feline sarcoma-leukemia viruses, murine leukemia virus, avian sarcoma virus, and visna virus. The genome of feline sarcoma-leukemia viruses, like that of RD-114 virus, is at least partly homologous to DNA from normal cats, suggesting that normal cats harbor endogenous genes coding for components of at least two classes of C-type viruses.     

Host range of mink cell focus-inducing viruses

The species host range of the recombinant, mink cell focus-inducing (MCF) class of murine retroviruses was determined in vitro and compared to the host range properties of xenotropic and amphotropic murine viruses. In contrast to xenotropic and amphotropic viruses, MCF viruses were restricted in the number of mammalian species they would infect. Cell lines from mouse, rat, mink, ferret, and cat were susceptible to MCF infection and certain virus isolates could infect rabbit cells, but cells from Chinese hamster, buffalo, bat, dog, monkey, and human were resistant to infection by most MCF viruses. The resistance of some of the latter cells was abrogated by phenotypic mixing with xenotropic virus, which demonstrated that MCF species host range was mediated by virus envelope-cell surface interaction. The host range uniformity of the various MCF isolates and the unique species distribution of sensitivity are consistent with the conclusion from other evidence that the MCF viruses comprise a class distinct from xenotropic and amphotropic viruses.     

Intracellular RNA complementary to the RNA genome of the Moloney-murine sarcoma virus complex

Intracellular RNA complementary to genomic RNA of the Moloney-murine sarcoma virus complex was detected in virus-producing rat cells. Hybrids formed between this novel type of RNA and its homologous viral genomic RNA were very stable exhibiting a T_m of 88°. Further characterization of the virus-specific complementary RNA revealed that it represented a minimal 74% of the viral RNA genome. Cross-hybridization data of hybrids formed between this viral complementary RNA and normal rat liver RNA or genomic RNA of several other mammalian retroviruses demonstrated its virus specificity and sequence relatedness. An examination of total cellular RNA from virus-producing cells for the number of intracellular virus-specific RNA copies disclosed 14 copies of viral complementary RNA per cell and 1272 copies of viral genomic RNA per cell.     

Specific sequences of the env gene determine the host range of two XC-negative viruses of the Rauscher virus complex

Two viruses which do not give rise to XC plaques in the standard XC assay (XC-negative) have been isolated from the Rauscher virus (RV) complex. These viruses differ in their host range. One, R-MCF-1, is dualtropic and will therefore infect both murine and non-murine cells. However, unlike other mink cell focus-inducing (MCF) viruses, it cannot infect NIH 3T3 cells. The other, R-XC-, is ecotropic. It will infect murine cells, including NIH 3T3 cells, but does not infect mink lung cells. Analysis of hybrid viruses, in which homologous regions of the genomes of R-MCF-1 and R-XC- virus were exchanged, indicated that the NH2-terminal portion of the gp70 is responsible for the particular host ranges of these viruses. The nucleotide sequence of the env gene of R-XC- virus was therefore determined and compared with the known env sequences of ecotropic MLVs and dualtropic MCF viruses of the Rauscher and Friend virus complexes. R-XC- virus was found to be a recombinant virus. Its env gene contained sequences derived from an endogenous env gene which were closely related to those of the MCF viruses but differed from any previously described sequences. The particular properties of R-MCF-1 and R-XC- virus suggest that the two viruses arose by recombination between R-MLV and two endogenous env sequences which differ from those of the known MCF viruses. If so, this suggests that the mouse genome contains at least five env sequences which can give rise to MCF-like viruses. In addition, since the host range and interference properties of R-XC- virus are very similar to those of the previously described ecotropic recombinant viruses, it may be that the ecotropic recombinant viruses arose by recombination with the same endogenous env sequences as did R-XC- virus.     

Identification of putative endogenous proviral templates for progenitor mink cell focus-forming (MCF) MuLV-related RNAs

Murine leukemia virus (MuLV)-related RNAs exhibiting different env deletions are believed to participate in the generation of leukemogenic mink cell focus-forming (MCF) viruses. We have cloned an endogenous MuLV provirus from AKR/J mouse DNA, designated as A-2, which may serve as template for the env-deleted E2 MuLV RNA, expressed in GIX+ mice (D.E. Levy et al., J. Virol. 56, 691-700 (1985]. We have also isolated an endogenous MCF-related DNA, A-1, which shared close sequence homology with the 7.2-kb RNA expressed in AKR mice (F. Laigret et al., J. Virol. 62, 376-386 (1988] and sustained an identical env deletion. The data indicate that putative precursor MCF-related RNAs are transcribed from a heterogenous family of env-deleted endogenous MuLV DNAs.     

The evolution of baboon endogenous type C virus: related sequences in the DNA of distant species

The cellular DNA of species distantly related to the baboon have been tested for the presence of sequences related to baboon endogenous type C virus. Hybrids between viral cDNA and cellular DNA were detected using very low stringency hydroxyapatite conditions. The results confirm and extend the observation that the related sequences are more conserved in African primates than in non-African primates. Our data suggest that this result is due to unusual conservation of viral sequences in the African primates. Our assay is sufficiently sensitive to detect distantly related sequences in mouse type C viruses and in other primate endogenous viruses, including both the type C virus isolated from macaques (MAC-1) and the type D virus isolated from langurs (LAD-1). However, using baboon viral cDNA, we cannot distinguish human DNA from the DNA of several non-primate mammalian species.     

Malignant transformation of rat embryo cells by a herpesvirus isolated from L2C guinea pig leukemia

A guinea pig herpesvirus (GPHV) was isolated from mink lung cells after cocultivation with L₂C leukemic cells. The virus was found to have a relatively narrow host range. The isolate was serologically related to the GPHV isolated by G. D. Hsiung and L. S. Kaplow [(1969). J. Virol. 3, 355–357] but not to the guinea pig cytomegalovirus as determined by neutralization tests. Complement-fixing antigen for human herpes simplex type 1 virus was detected in cell suspensions prepared from GPHV-infected mink cells. Rat embryo cells infected with a GPHV mink isolate showed morphological transformation. The transformed cells produced neither infectious virus nor virus-specific antigens detectable by either complement fixation or fluorescent antibody tests. Virus particles were not detected in the transformed cells. No infectious GPHV could be rescued from the transformed cells by cocultivation with guinea pig cells. The morphologically transformed cells formed large cell aggregates and grew in this aggregate form when suspended in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities while the normal rat embryo cells did not. The transformed cells produced tumors when transplanted subcutaneously into newborn rats. Herpesvirus complement-fixing antigen was detected in rat tumor cells but infectious virus was not recovered from rat tumor cells.     

Identification of a normal vertebrate cell protein related to the p21 src of Harvey murine sarcoma virus

Harvey murine sarcoma virus (Ha-MSV) is a recombinant virus between genetic sequences of a helper-independent mouse type C virus and rat nucleic acid sequences. The rat sequences of Ha-MSV encode a 21,000-dalton protein (p21) which is responsible for the maintenance of the virus-induced transformation. Employing rat antisera containing antibodies directed against Ha-MSV p21, we have detected lower amounts of a 21,000-dalton cellular homolog in uninfected cells derived from a variety of vertebrate species including rat, mouse, mink, hamster, rabbit, turkey, bat, cat, dog, horse, monkey, and human. Although we could detect with the same antisera a phosphorylated form of the viral p21, we were unable to immunoprecipitate a phosphorylated form of the endogenous p21. Partial proteolytic peptide mapping by Staphylococcus aureus V8 protease demonstrated that the Ha-MSV p21 is structurally very similar to the endogenous cellular homolog which is itself chemically highly conserved in a variety of vertebrate cells. The present data suggest that a normal cellular gene encoding an antigenically and chemically related protein of the Ha-MSV p21 is present in many vertebrate species.