Robin A. Weiss

Virus infection of murine teratocarcinoma stem cell lines

Abstract Three clonal murine teratocarcinoma stem cell lines were studied for susceptibility to infection by a variety of different viruses. When in the undifferentiated state, these embryonal carcinoma stem cells are as sensitive to infection by encephalomyocarditis, Sindbis, vaccinia and vesicular stomatitis viruses as are embryo fibroblasts derived from the same mouse strain. The undifferentiated teratocarcinoma cells, however, unlike the fibroblasts, are entirely refractory to infection with murine leukemia virus. If the stem cells are allowed to differentiate to form a variety of differentiated cell types, they become permissive for murine leukemia virus replication at a low level. Several techniques were used to elucidate the block to murine leukemia virus infection. In the one nullipotent cell line which does not differentiate in vivo or in vitro, virus adsorption and penetration are not restricted (as measured by infection with murine leukemia virus pseudotypes of vesicular stomatitis virus), but an integrated proviral DNA copy cannot be detected. In a pluripotent stem cell line, proviral DNA sequences are detected, but neither transcription into viral specific RNA nor viral specific protein synthesis is observed. These findings suggest that control of murine leukemia virus replication in the cells is a function of the stage of differentiation and perhaps also of the genetic composition of the teratocarcinoma stem cells.

The distribution of endogenous chicken retrovirus sequences in the DNA of galliform birds does not coincide with avian phylogenetic relationships.

The chicken is a domesticated form of Red Jungle-fowl (Gallus gallus), which belongs to the Pheasant family (Phasianidae) within the order Galliformes. Domestic chickens carry the genome of the endogenous retrovirus RAV-O as DNA sequences integrated into host chromosomes transmitted through the germ line. We have examined the presence and distribution of RAV-O-related sequences in the DNA of Red Junglefowl and other closely related species of Junglefowl, as well as more distantly related Pheasants and Quail. DNA sequences homologous to RAV-O were analyzed by molecular hybridization in liquid and after electrophoresis of restriction endonuclease fragments. The presence of RAV-O-related sequences in avian DNA does not correlate with phylogenetic relationships. Under stringent conditions of hybridization in liquid, DNA sequences homologous to RAV-O cDNA were detected at high levels (greater than 80% homology( only in the genomes of the domestic chicken and its phylogenetic ancestor, the Red Junglefowl (Gallus gallus). The DNA of two other species of Gallus (G. sonnerati, Sonnerats Junglefowl and G. varius, Green Junglefowl), of Ring-necked Pheasant and of Japanese Quail contained sequences with less than 10% homology to RAV-O cDNA. Under conditions permitting mismatching, however, Ring-necked Pheasant DNA hybridized up to 50% of the RAV-O cDNA, and Quail DNA 24%, whereas the extent of hybridization to Sonnerats and Green Junglefowl DNA was not markedly increased. Analysis of restriction enzyme digests revealed several distinct fragments of DNA hybridizing to chick retrovirus cDNA in both Red Junglefowl and domestic chicken, and multiple fragments in DNA from two species of Phasianus. No fragments with sequences related to chicken retroviruses were found, however, in digests of DNA prepared from Sonnerats, Ceylonese and Green Junglefowl, from two other Pheasant genera (Chrysolophus and Lophura), or from one Quail genus (Coturnix). Thus the DNA of three Junglefowl species closely related to Gallus gallus lacked RAV-O sequences while the DNA of more distantly related Phasianus species showed significant homology. These results show that RAV-O-related sequences have not diverged together with the normal host genes during the evolution of the Phasianidae. Although RAV-O sequences are endogenous in all domestic chickens and Red Junglefowl studied thus far, it appears that the RAV-O genome has been introduced relatively recently into the germ line of Gallus gallus, following speciation but before domestication, and independently of the related sequences found in members of the genus Phasianus.

Pseudotypes of avian sarcoma viruses with the envelope properties of vesicular stomatitis virus.

Upon superinfection of cells producing Rous sarcoma virus (RSV) with temperature-sensitive mutants of vesicular stomatitis virus (VSV), two kinds of pseudotype viruses are produced: VSV genomes within particles bearing the envelope antigens of RSV, denoted VSV(RSV), and RSV genomes within particles bearing the envelope antigens of VSV, denoted RSV(VSV). The VSV(RSV) pseudotypes are recognized as the fraction of plaque-forming units resistant to neutralization by antiserum to VSV or, in the case of thermolabile envelope mutants of VSV, resistant to heat inactivation; they possess the host range restrictions of RSV and are neutralized by antisera specific to the RSV subgroup. The RSV(VSV) pseudotypes are recognized as the fraction of focus-forming units which transforms chick cells resistant to infection with the strain of RSV used. Both kinds of pseudotypes are produced concomitantly with VSV synthesis. VSV(RSV) particles comprise up to 12% of the VSV progeny titer and RSV(VSV) up to 1% of the RSV titer, but pseudotype fractions varied according to the VSV mutant used for superinfection. The proportions of pseudotypes in harvests of mixed infections are not reduced by filtration through 0.2-μm pore size filters to eliminate large aggregates of virus particles, and pseudotypes are not formed by mixing pure-grown RSV and VSV particles in vitro. VSV acts as a helper virus for BH-RSV(-), which is defective in envelope antigen, but not for αBH-RSV(-), which is also defective in RNA-directed DNA polymerase activity. The titer of BH-RSV(VSV) is enhanced by the presence of the avian leukosis helper virus, RAV-1, and more than 90% of this mixed pseudotype stock is neutralized by antiserum to either VSV or RAV-1, indicating that the RSV particles bear a mosaic of both VSV and RAV-1 envelope antigens. RSV(VSV) pseudotypes transform cells of four out of five mammalian species tested. Like RSV of subgroup D and B77, the focus-forming titer of RSV(VSV) assayed on mammalian cells is 1000-fold lower than on chick cells.

Pseudotypes of vesicular stomatitis virus determined by exogenous and endogenous avian RNA tumor viruses.

Abstract Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of avian RNA tumor viruses. The VSV pseudotypes possess the specific host range, interference, and antigenic properties of the tumor virus. Growth of VSV in cells expressing chick cell-associated helper factor coded by an endogenous viral genome yields pseudotypes with the envelope specificity of the helper factor.

Phenotypic mixing between avian and mammalian RNA Tumor viruses: I. envelope pseudotypes of rous sarcoma virus

Abstract Pseudotypes of Rous sarcoma virus (RSV) with the envelope properties of six strains of mammalian leukemia viruses (MaLV) were produced after mixed infection in avian cells permissive for the replication of both types of virus. RSV pseudotypes with the envelope antigens of xenotropic and ecotropic murine leukemia virus were also obtained by fusion of mammalian cells producing leukemia virus with avian cells producing RSV, but RSV(MaLV) pseudotypes were not obtained by MaLV superinfection of RSV-transformed mammalian nonproducer cells. Functional RSV(MaLV) pseudotypes were obtained with both nondefective RSV and RSV defective in envelope antigens, but were not obtained with RSV which is defective in RNA-directed DNA polymerase.

Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus

Abstract Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.

Assembly of membrane glycoproteins studied by phenotypic mixing between mutants of vesicular stomatitis virus and retroviruses

Abstract Temperature sensitive ( ts ) mutations of vesicular stomatitis virus (VSV), Indiana serotype, which belong to complementation group V ( ts V) have been shown to affect the viral envelope glycoprotein, or G protein. When ts V mutants are grown in cells producing avian leukosis viruses, the titers of infectious VSV obtained at the nonpermissive temperature are 10 4 -fold higher than in control cells. Cells releasing murine leukemia viruses or avian reticuloendotheliosis virus rescue VSV ts V mutants much less efficiently. The rescued virions have the properties of envelope pseudotypes in that their host range is restricted to that of the helper retrovirus, they are neutralized by anti-retrovirus antibodies but not anti-VSV antibodies, and they are not thermolabile. Sensitive serological techniques, including the use of complement-mediated virolysis, immunoprecipitation, and monoclonal antibody reacting with G protein, show that VSV pseudotypes produced at the nonpermissive temperature have no detectable G protein, whereas VSV particles released from retrovirus infected cells at the permissive temperature have mosaic envelopes bearing both VSV G protein and retrovirus glycoprotein. In mixed infections of Rous sarcoma virus (RSV) and VSV ts V mutants, pseudotype particles with RSV genomes and VSV envelope antigens are produced only at the permissive temperature. In contrast, substantial yields of RSV(VSV) pseudotypes but no VSV(RSV) pesudotypes are obtained at the nonpermissive temperature with VSV carrying mutations in complementation group III, which affect M protein. Thermolabile VSV ts V mutants form RSV(VSV) pseudotypes which also are thermolabile. The kinetics of heat inactivation of G protein function in ts V mutants is the same in VSV particles with unmixed envelopes and with mosaic envelopes. From these studies of phenotypic mixing we draw the following conclusions: (i) The synthesis of functional M protein but not G protein is essential for the maturation of VSV virions. (ii) VSV M protein is not required for the assembly of G protein into retrovirus virions. (iii) The thermolabile nature of ts V VSV mutants is an intrinsic property of the G protein, independent of the type of virion into which it is incorporated and of other viral glycoproteins which may be assembled into the envelope of the same virion.

Heterophil human antibodies recognize oncovirus envelope antigens: Epidemiological parameters and immunological specificity of the reaction

Abstract Human sera were previously shown to possess antibodies capable of recognizing purified retrovirus envelope glycoproteins in standard radioimmunoprecipitation assays (RIAs). In a logical extension of the earlier studies, epidemiological characteristics and the exact specificity of the human antiviral antibodies was analyzed. The antiviral antibodies are not detectable in cord sera but show an age-dependent peak in early childhood and subsequently persist lifelong. Based on epidemiological and additional data obtained by serurn absorptions to cell surfaces, competition RIAs with animal sera and experiments employing deglycosylated virus envelope antigens, we conclude that the majority (if not all) of normal human sera contain naturally occurring, heterophil antibodies that react with the carbohydrate moieties of retrovirus envelope antigens. Similar conclusions have independently been reached by M. Barbacid, D. Bolognesi, and S. A. Aaronson (1980, Proc. Nat. Acad. Sci. USA 77 , 1617–1621) and by H. W. Snyder and E. Fleissner (1980, Proc. Nat. Acad. Sci. USA 77 , 1622–1626).

Differences between the endogenous and exogenous dna sequences of rous-associated virus-O

DNA sequences related to the endogenous retrovirus of chickens, Rous-associated virus-O (RAV-O), have been examined using site-specific DNA endonuclease analysis of cellular DNA derived from line 15 and line 100 chickens. Individual embryos from both inbred lines were used as a source of embryonic fibroblasts from which cellular DNA was isolated. Analysis of DNA containing either endogenous RAV-O sequences alone or both endogenous and exogenous RAV-O sequences produced identical patterns of RAV-O-specific DNA fragments after digestion with the endonucleases Eco RI, Hind III, BgI II, Bam HI or Xho I. Similar analysis with endonucleases Hinc II or Hha I, however, produced several RAV-O-specific DNA fragments which were derived from cellular DNA containing both endogenous and exogenous RAV-O sequences but not from cellular DNA containing only endogenous sequences. Although some differences exist between the DNA fragments specific for the endogenous viral sequences of line 15 and line 100 cellular DNA, the DNA fragments specific for the exogenous viral sequences were identical between the two inbred lines. Cleavage of an unintegrated linear RAV-O DNA molecule with Hinc II or Hha I produced DNA fragments identical to those specific for the exogenously acquired RAV-O provirus. This suggests that these characteristic fragments contain no cellular DNA. The potential DNA junction fragments containing both viral and cellular DNA, identified after analysis of DNA that contains both endogenous and exogenous viral sequences, were identical to those observed after analysis of DNA containing only endogenous viral sequences. These results support the following conclusions. First, exogenous proviral sequences are integrated into chicken cell DNA following an interaction between viral and cellular DNA that is specific with respect to the virus and nonspecific with respect to the cell. Second, both the free linear RAV-O DNA intermediate and the newly integrated exogenous provirus contain specific endonuclease sites that are not found in endogenous RAV-O DNA sequences. These results suggest that the formation of the exogenous DNA provirus involves specific alteration of the endogenous viral DNA sequences before reinsertion of the sequences as the exogenous RAV-O DNA provirus. It is possible that newly integrated exogenous RAV-O sequences are characterized by specific differences in the pattern of base methylation and a limited sequence arrangement.

Chronic Infections of C-Type RNA Viruses

Oncornaviruses comprise a large group of structurally similar viruses, some of which are known to cause malignant disease in their hosts. They are morphologically classified into B-type virus particles, characteristic of the murine mammary carcinoma virus, and C-type viruses, characteristic of widespread infections of numerous species. While not all C-type virus strains are known to be pathogenic, they are frequently associated with lymphoma, leukemia and other mesenchymal tumors. The viral etiology of these kinds of tumor in the domestic fowl was first noted nearly 70 years ago when Ellerman and Bang in 1908 demonstrated the infectious transmission of leukosis by cell-free filtrates and Rous in 1911 made similar observations with sarcomas. Nevertheless, 40 years elapsed before Gross was able to prove that thymic lymphoma in the AKR mouse was similarly caused by a filterable virus, and only in recent years has it become evident that C-type viruses are also a major cause of hemopoietic neoplasms in other rodents and in cats, cattle and possibly gibbons too. C-type viruses have now been isolated from numerous species of mammals, birds and at least one reptile (the viper). We expect that many more species of virus remain to be discovered, and that the spectrum of disease with which they are associated will not be confined to neoplasia. The association of a murine C-type virus with the “autoimmune” hemolytic anemia and glomerulonephritis in New Zealand mice (15), and the presence of C-type virus genetic information in latent or partially expressed states in so many vertebrate species, suggests that C-type viruses might be implicated in some chronic inflammatory diseases.