Spontaneous expression of endogenous type C RNA virus by BALB/c splenic B lymphocytes in continuous culture.

B-lymphocyte cultures were established from spleens of BALB/c, C57B1/6, NIH Swiss, and SWR mice of various age groups. Spontaneous, consistent, and thus predictable release of a B-tropic mouse endogenous virus occurred from the very first passage in cultured lymphocytes derived from BALB/c mice 6 months old or older but not from similar lymphocytes derived from BALB/c mice of 1.5 or 3 months of age. C57Bl/6, NIH Swiss, and SWR mice belonging to various age groups ranging between 1.5 and 18 months failed to exhibit such a spontaneous release of viral particles. We conclude that in BALB/c splenic B lymphocytes a breakdown of cellular control mechanisms occurs in older animals leading to viral production while such a phenomenon is absent in C57Bl/6, NIH Swiss, and SWR mice.     

Activation of endogenous type C virus by amino acid alcohols.

Amino acid alcohols were examined for their ability to inhibit protein synthesis and induce endogenous type C virus from Kirsten sarcoma virus (KiSV)-transformed Balb/c mouse cells. Histidinol and tyrosinol, amino alcohols of histidine and tyrosine, were very effective short-term activators of virus. Both inducers were very efficient inhibitors of protein synthesis reducing [³H]leucine incorporation by more than 90% in 1 hr. Two other alcohols, valinol and methioninol, also reduced protein synthesis but gave low activation. The activated virus had the host range of the xenotropic Balb virus:2, and following removal of inducer, the activated state decayed rapidly.     

The polypeptides induced in Drosophila cells by Drosophila C virus (strain Ouarzazate)

The Ouarzazate strain of Drosophila virus (DCV0) was grown in Drosophila melanogaster tissue culture cells, and [35S]methionine-labeled virions were found to contain a group of major structural proteins with a molecular weight of approximately 30,000 as well as several minor proteins of higher molecular weight and a protein of approximately 10,000 daltons. Using a range of pulses, chases and gel systems, examination of the intracellular proteins induced by DCV0 showed the presence of 17 polypeptides not found in uninfected cells. The synthesis of virus-induced polypeptides was extremely asymmetric with a rapid appearance of the major virus structural proteins and a much slower appearance of the lowest molecular weight structural protein (VP4). Processing of virus-induced proteins including the appearance of VP4 was demonstrated using pulse-chase after pulsing with [35S]methionine. While the highest molecular weight induced protein found in infected cells was 146,000, pretreatment of cells with iodoacetamide resulted in the appearance of a protein with a molecular weight of approximately 200,000. The evidence presented in this paper supports the inclusion of DCV0 in the Picornaviridae group.     

Factors determining the susceptibility of NIH swiss mice to erythroleukemia induced by Friend murine leukemia virus

The phage φ80, an Escherichia coli temperate phage, has similarities in sequence and physiology with bacteriophage λ. The isolation of new λ derivatives which carry recombination genes of φ80 (red₈₀ genes) under the control of the λ immunity region is described. Study of these hybrids suggests that the recombination system of φ80 is analogous to the recE system of Escherichia coli. Expression of φ80 recombination in lysogens corrects, like recE, the recombination defect presented by recB^? mutants. The φ80 recombination system also promotes phage recombination in recA hosts, which is also a characteristic of the recE system. The study of recombination defective red₈₀ phages suggests that the φ80 recombination system also determines φ80 growth on P2 lysogens.     

Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells tranformed by Kirsten or Harvey murine sarcoma virus.

A similar protein of 21,000 MW (p21) coded for by Harvey or Kirsten murine sarcoma virus has been identified in nonproducer cells transformed by these two viruses. Antisera prepared from rats bearing tumors induced by syngeneic transplantation of NRK cells transformed by Harvey murine sarcoma virus (Ha-MuSV) specifically precipitated the Ha-MuSV p21 from a nonproducer Balb/c mouse cell and a nonproducer dog cell transformed by HaMuSV. The same antisera also precipitated a similar protein, Ki-MuSV p21, from a nonproducer mink cell transformed by Kirsten murine sarcoma virus (Ki-MuSV). Both the p21 of Ha-MuSV and of Ki-MuSV are phosphoproteins. Previous studies have reported a virus-specific p21 polypeptide from translation of Ha-MuSV RNA in cell-free protein synthesis systems (W. P. Parks and E. M. Scolnick, 1977, J. Virol.22, 711–719; T. Y. Shih, D. R. Williams, M. O. Weeks, J. M. Maryak, W. C. Vass, and E. M. Scolnick, 1978, J. Virol.27, 45–55). This p21 protein was specifically precipitated by the same anti-tumor sera. Similarly, a p21 polypeptide translated from Ki-MuSV RNA was also specifically precipitated by the antitumor sera. Therefore, it is concluded that the p21 of Ha-MuSV and Ki-MuSV are homologous proteins coded for by homologous sequences found in the recombinant genomes of Ha-MuSV and Ki-MuSV.     

Increase in the saturation of C18 fatty acids induced by coxsackie B6 virus in vero cells

There is an increase in the saturated 18-carbon fatty acid (C18FA) in Vero cells infected with Coxsackie B6 virus. The increase is reversible with a peak at 4 hr postinfection, and is proportional to virus input (m.o.i.). The appearance of the cytopathic effect (CPE) is coincidental with the increase in the saturation of C18FA, and is also related to increase in cell leakiness as measured by ¹⁵Cr release. Replacement of the overlay medium with fresh medium results in a further twofold increase in saturated C18FA. The overlay medium of the infected cells contains a factor which, in uninfected cells, produced an increase in unsaturated C18FA. The causal relationship of the changes in the saturation of the C18FA and the CPE and cell lysis is discussed.     

Lack of interferon-induced transfer of viral resistance against murine leukemia virus or poliovirus in cocultivated cells

It has recently been reported that there can be a transfer of the interferon (IFN)-induced viral resistance between cocultivated animal cells. However, in those systems the challenge virus was capable of replicating in both cell types. A more convincing conclusion could be obtained if the infecting virus could not replicate in the cell type which is homologous to the species of IFN that is added to the cultures. An inhibition of the yield of this virus would indicate that there was a transfer of the antiviral state from one cell species to another. In cocultivated mouse and human cells, a murine ecotropic type-C virus can only multiply in the mouse cells whereas poliovirus will only replicate efficiently in the human cells. It is clearly evident from this study that only homologous but not heterologous fibroblast IFN is capable of inhibiting the replication of either of these viruses in various combinations of cocultivated cells.     

Organization and expression of endogenous murine mammary tumor virus genes in mice congenic at the H-2 complex

The organization and expression of endogenous murine mammary tumor virus (MuMTV) genetic information was examined in a number of mouse strains Congenic at the major histocompatibility complex (MHC). Analysis of restriction endonuclease-generated fragments of cellular DNA revealed no differences in the integration sites (EcoRI)or internal cleavage sites (PstI) of MuMTV proviruses in any of the (C7BL/10 (B10) Congenic strains tested. Similar studies indicated that the proviruses of BALB/c and BALB.B mice are indistinguishable, and that the restriction endonuclease-generated fragments of BALB/c mice are similar to those of the B10 strain. These proviruses behave as stable genetic elements which have been well conserved during the derivation of the Congenic strains. Also, since none of the characteristic MuMTV proviruses of the donor strains could be identified in the Congenic mice, they do not seem to be closely linked to the H-2 complex. When concentrations of viral RNA in lactating mammary glands (LMGs) were measured by molecular hybridization kinetics, comparable levels of MuMTV RNA were found in all the B10 Congenic mice; considerably less viral RNA was detected in mice with a BALB/c background. This viral RNA in mammary gland cells was not translated into detectable gp49, the major MuMTV glycoprotein. These results indicate that the differences observed in susceptibility to mammary tumorigenesis induced in H-2 Congenic mice by exogenous MuMTV are not influenced by the organization or expression of endogenous MuMTV proviral genes.     

Preferential expression of endogenous type C viral antigen in Rhesus placenta during ontogenesis

A radioimmunoassay for the major internal structural protein (p26) from Macaca arctoides type C virus (MAC-1) was used to score antigen expression in rhesus placenta and other fetal organs obtained at various times of gestation. Antigen expression was detected in 16 out of 16 placental specimens but not in fetal heart, lung, spleen, liver, kidney, intestines, skeletal muscle, testes, and endometrial scrapings of decidua from eight selected animals, nor in thymus from two animals. The levels of antigen detected in placenta ranged between 2 and 218 ng/mg protein with a correlation between lower antigen expression and term gestation or parity greater than 10. A 10-fold higher level of antigen expression was detected at the external surface of placenta near decidua than in the remainder of the placenta toward the amniotic surface. Thus, even within the placenta, there was a preferred site for endogenous retroviral antigen expression. Last, substantial interindividual variation exists in regard to the amount of placental p26 expression in preterm animals of low or intermediate parity.     

Regulation of endogenous type C virus expression during normal myeloid cell differentiation. Evidence for a role in promoting myeloid cell proliferation and differentiation

A specific regulatory pattern for the expression of endogenous type C virus was found during differentiation of normal myeloblasts from different strains of mice. Endogenous virus was expressed in the myeloblasts. Upon induction of differentiation by the normal macrophage- and granulocyte-inducing protein MGI, there was a rapid enhancement of virus production followed by an inhibition of virus secretion and an intracellular accumulation of viral proteins. There was finally a complete shutoff of viral protein synthesis at the terminal stage of differentiation, so that the mature cells no longer expressed viral proteins. The comparison of normal and fully differentiatable MGI⁺D⁺ leukemic myeloblasts has shown that the regulatory pattern for virus production during differentiation in the normal myeloblasts was similar to that during differentiation in the leukemic myeloblasts. Ecotropic and xenotropic viruses were detected in the normal myeloblasts, whereas only ecotropic virus was detected in the leukemic myeloblasts. Only the production of ecotropic virus was enhanced upon induction of differentiation of the normal myeloblasts by MGI, whereas there was a shutoff of both xeno- and ecotropic viruses at the terminal stage of differentiation. Dexamethasone, lipopolysaccharide, dimethylsulfoxide, and low concentrations of actinomycin D increased the MGI-induced enhancement of ecotropic virus in both the normal and the leukemic myeloblasts, but did not affect the production of the xenotropic virus. Superinfection of the normal myeloblasts with high titers of their own endogenous ecotropic virus, as in the case of infection with virus from MGI⁺D⁺ leukemic cells, increased the proliferation of myeloblasts, and in the presence of MGI this then resulted in an increased number of mature cells. It is concluded that the observed pattern for endogenous ecotropic type C virus expression is part of the developmental program of mouse myeloid cell differentiation and that endogenous type C viruses may serve as normal physiological agents for the promotion of myeloblast cell proliferation, which in the presence of the normal inducer of differentiation then results in an increased number of differentiated cells.     

Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies

Monoclonal antibodies produced by hybrid cells in culture are chemically homogeneous reagents that react with constant avidity to single antigenic determinants (epitopes). As such, these antibodies are remarkably specific probes for small regions of complex antigenic proteins. We have used a panel of monoclonal antibodies in competition binding assays to investigate the arrangement of six epitopes on the envelope proteins of AKR leukemia virus. It was reasoned that if two epitopes were adjacent to each other on a single protein, the binding of antibody to one epitope would sterically hinder the binding of antibody to the other epitope. On the other hand, if the two epitopes were at distant sites on the protein, the binding of antibody to one epitope would not influence the binding of antibody to the other epitope. The results of these competition binding assays demonstrated the presence of two distinct antigen sites on both the gp70 and p15(E) envelope proteins. With the gp70 protein, one antigen site contained the gp70^b and gp70^c epitopes; the other antigen site on this protein contained the gp70^a epitope. With p15(E), one of the antigen sites contained the p15(E)^b and p15(E)^c epitopes, while the other site contained the p15(E)^a epitope. These findings demonstrate the utility of this type of serological analysis for the study of the tertiary structure of individual viral proteins.     

The effect of ethidium bromide on C type virus production and induction.

Ethidium bromide suppresses induction by iododeoxyuridine (IUDR) of mouse xenotropic type C viruses from BALB S + L-cells. This effect can be observed up to 24 hr after treatment with IUDR. The dye also reduces infectious virus production by chronically infected cells. These results indicate that EB can affect type C virus production by cells already containing integrated provirus.     

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.     

Characterisation of murine pneumonia virus proteins.

Twelve [³⁵S]methionine-labelled polypeptides ranging in molecular weight from 12,000 to 191,000, which were not present in mock-infected BS-C-1 cells, were detected in cells infected with murine pneumonia virus (PVM). All but two of these polypeptides were present in cell-released virus concentrated by polyethylene glycol precipitation and partially purified by equilibrium centrifugation. Two major glycopolypeptides, VPII (79,000 mw) and VPIII (68,000 mw) were present in partially purified virus but only VPII was prominent intracellularly. Nucleocapsids were isolated from lysates of infected cells by centrifugation in a gradient of 15–50% metrizamide and contained a major (VPIV) and a minor (VPVI) polypeptide with molecular weights of 42,300 and 35,700, respectively. Polypeptides with the electrophoretic mobilities of six of the PVM polypeptides (VPIV, VPV, VPVII, VPIX, VPX, and VPXI) were detected by in vitro translation of cytoplasmic RNA from PVM-infected cells in a cell-free system prepared from rabbit reticulocytes. PVM resembled RS virus, the other member of the pneumovirus genus, in its ability to multiply in enucleate cells. It is distinguished, however, by its four low molecular weight polypeptides (VPIX, VPX, VPXI, and VPXII), which appear to be primary gene products, and by the different electrophoretic mobility of the glycopolypeptides (VPII and VPIII).     

Syncytium formation induced by baboon endogenous virus in several transformed human cell lines

Several types of cultured human cells derived from malignant tumors and transformedin vitro by DNA tumor viruses. RNA tumor viruses, chemical carcinogens, or⁶⁰Co γ radiation were fused into syncytia when cocultivated with baboon endogenous virus producing human embryonic cells (BaEV-HEC). On the other hand, neither diploid human cell lines nor cells from normal human embryos were fused when cocultivated with BaEV-HEC. Thus, syncytium formation induced by BaEV is dependent upon transformation of human indicator cells rather than upon transforming agents themselves. Concentrated cell-free BaEV suspensions also induced syncytia in human indicator cells within 2 hr after inoculation. The presence of cycloheximide in culture medium had no effect on early syncytium formation. Human indicator cells exposed to low concentrations of BaEV did not form syncytia but produced the virus. These findings strongly suggest that cell fusion induced by BaEV is fusion from without. Specific antiserum against BaEV (M7) blocked this syncytium formation but did not block cell fusion mediated by Mason-Pfizer monkey virus or simian sarcoma virus type I. These observations indicate that the syncytium formation is BaEV specific. The findings in this study suggest that syncytium formation induced by BaEV is a specific characteristic of malignant or transformed human cells.     

Expression and disposition of the murine mammary tumor virus (MuMTV) envelope gene products by murine mammary tumor cells

Three murine mammary tumor virus (MuMTV)-producing epithelial cell lines derived from murine mammary tumors were examined in order to identify the MuMTV-specific cell surface antigens and their distribution on the cell surface, to study the kinetics of the MuMTV envelope precursor processing, virus assembly, and release, and to characterize the soluble MuMTV antigens that are shed into culture medium. Cell surface labeling experiments showed that only the mature MuMTV envelope glycoproteins gp52 and gp36 were exposed on the cell surface, and that gp52 was more abundant than gp36. In cells producing large quantities of MuMTV, expression of gp52 on the cell surface was shown by immunoelectron microscopy to be localized predominantly on the surface of budding virions and not on smooth areas of the cell surface where virus was not budding. The cell surface associated gp36 was found not to be incorporated into budding virions. A few cells in all three cell lines were found to produce only a few or no MuMTV particles and in these cells, unlike in the high virus-producing cells, considerable quantities of gp52 were expressed on the surface membrane. All three cell lines were found to shed large amounts of the MuMTV env precursor polyprotein as well as the mature non-virion-associated glycoprotein, gp52, into the culture medium. The envelope precursor protein (P75^env) that was shed into the culture medium was found to differ from the predominant form of the cellular env precursor Pr70^env in that (1) P75^env migrated with an apparent higher molecular weight than Pr70^env in SDS gels; (2) Pr70^env contained only the core oligosaccharide, whereas P75^env contained fucose in addition to the core sugars; (3) two-dimensional gel electrophoretic analysis showed that Pr70^env could be resolved into three to four components migrating in the basic region of the isoelectric focussing gel (pH 7–8), whereas P75^env was resolved into 9–13 components migrating in a more acidic region of the gel (pH 5–7). The molecular structure of the exfoliated gp52 was found to be similar to that of the gp52 that was incorporated into the virions although the virion-associated gp52 was not the source of the gp52 in the medium. Our quantitative pulse-chase studies suggest that of the two populations of MuMTV env precursors that are present in MuMTV-producing cells, only Pr70^env is processed intracellularly to give rise to the mature MuMTV envelope proteins gp52 and gp36.     

Partial characterization of a Moloney murine sarcoma virus 85,000-dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus

The viral proteins specified by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in its transforming gene were examined. Normal rat kidney cells infected with this replication-defective virus have transformed cell characteristics at 33° but revert to a normal phenotype at 40°. At the temperature permissive for transformation, the cells contained an 85,000-dalton protein (P85) which had antigenic determinants of p15, pp12, and p30, and also tryptic peptides characteristic of p15 and p30 as well as additional unidentified tryptic peptides. P85 was only detectable at the permissive temperature. A 58,000-dalton protein (P58) was also detected. It had both antigenic determinants and tryptic peptides of p15, pp12, and p30. P58 was seen at both temperatures. Phosphorylation experiments indicated that P58 is a phosphoprotein whereas ³²P-labeled P85 was not observed. Temperature shift experiments showed that newly synthesized P85 was first detected between 2 and 3 hr following transfer of cultures to 33°. Morphological and biochemical changes indicative of transformation occurred 8 or more hr after temperature shift. These results are consistent with the interpretation that P85 contains peptide sequences derived from both the gag gene and the MuSV-acquired or src gene sequences.     

Morphologic changes in the rabbit SIRC cell line induced by simian sarcoma-associated virus

Rabbit cornea (SIRC) cells were altered morphologically upon simian sarcoma-associated virus infection. These focal alterations could be used for virus assay. Unusual small cytoplasmic particles of uncertain origin were observed by electron microscopy in addition to typical C-type particles.