Virus Group-specific Antigen Research Articles

Inhibition of rous sarcoma virus assembly by treatment with 2′,5′ adenosine nucleotides

We have investigated the influence of 2′,5′ adenosine nucleotides on the replication and transformation of cells by Rous sarcoma virus (RSV). Treatment with the nucleotides ppp2′,5′A 4 and 2′,5′A 4 causes a striking reduction (50-fold) in the yield of infectious progeny virus, while ppp2′,5′A 2 and 2′,5′A 3 had virtually no effect. The reduction in infectivity seen with 2′,5′A 4 nucleotides is paralleled by a smaller but significant (three- to four-fold) reduction in the amount of particles released as measured by reverse transcriptase activity and levels of viral structural proteins. The reduced infectivity of released particles is not due to viral RNA being missing since the amount of genomic RNA in particles from 2′,5′A 4-treated cultures was likewise only reduced by a factor of 2–3. Pulse-chase radioactive label experiments showed that processing of both viral group-specific antigens ( gag) and viral envelope glycoprotein (env) gene products was completely normal in nucleotide-treated cultures, but that the rate of appearance of viral proteins in mature virus in the culture supernatants was reduced by a factor of about 3–4. Taken together, the data show that assembly of viral structural proteins into virions which can be released into the medium is slowed, and that assembly of virus particles with reduced infectivity follows upon nucleotide treatment. This inhibition of infectious virus production takes place without significant toxic effects on the cell; host protein synthesis is only 20% inhibited. There is also no significant effect on the secretory ability of the cells as measured by total protein release into the medium or release of fibronectin. The transformed cell phenotype was also subtly affected by 2′,5′A 4, but not by other oligomers. Plasminogen activator protease activity was sharply reduced upon treatment, while other typical features of RSV-transformed cells such as elevated hexose transport, and pp60 src -associated protein phosphokinase activity, were little affected.

Fusion injection of Rous Sarcoma virus proteins into Rous sarcoma virus-transformed, non-producing hamster cells causes release of infectious virus.

Purified virus proteins from transformation-defective (td) mutants of Rous sarcoma virus PrA or PrB were trapped in human erythrocyte ghosts which, after resealing, were fusion-injected into hamster RBH cells or rat TWERC cells. These cell lines are non-productively transformed by subgroup C Rous sarcoma virus. After fusion injection the hamster RBH cells released transforming subgroup C Rous sarcoma virus. No infectious virus could be rescued from rat TWERC cells. Since previous experiments have shown that fusion injection of the purified Rous sarcoma virus protein p15 into hamster RBH cells caused cleavage of the precursor protein pr76 to form the virus group-specific antigen (gag) but did not induce infectious virus, we conclude that in addition to p15 other virus proteins are required to induce virus rescue in hamster RBH cells.

Analysis of avian leukosis virus infections with an enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for avian leukosis virus group-specific antigen was used to study infections with and shedding of avian leukosis virus in a commercial flock of chickens with a known high incidence of infection. Avian leukosis virus group-specific antigen was detected in serum or cloacal washings from 76% of a group of 100 61-week-old hens. With eggs collected during the next 3 weeks, antigen was detected in the albumen of 88% of the eggs from ELISA-positive hens and 2% of the eggs from ELISA-negative hens. Results of assays for infectivity correlated closely with the ELISA results. Serum and cloacal specimens were almost equally sensitive in detecting infection; however, a higher proportion of cloaca-positive hens (97%) than serum-positive hens (91%) shed virus in their eggs. Similar results were obtained from a second sampling of eggs taken when the hens were 84 weeks old, after an intervening molt. Avian leukosis virus infection was correlated with reduced egg production and increased mortality. Eggs were produced by 100% of the ELISA-negative birds during both sampling periods, whereas only 69% of the ELISA-positive birds produced eggs at 61 weeks and 76% produced eggs at 84 weeks. All birds that were ELISA negative at 61 weeks survived the experiment. Of 14 ELISA-positive birds that died and were examined postmortem between 61 and 84 weeks, 8 had malignancies, and the remainder died of a variety of nonmalignant diseases.

Infection of Feline Embryo Adherent Cells With Feline Leukemia Virus: Feline Oncornavirus-Associated Cell Membrane Antigen Expression and Morphologic Transformation<xref ref-type="fn" rid="fn2">2</xref>, <xref ref-type="fn" rid="fn3">3</xref>

Feline embryo adherent cells were infected with the Richard or Kawakami-Theilen strains of feline leukemia virus (FeLV) and examined for feline oncornavirus-associated cell membrane antigen (FOCMA), viral group-specific antigen (gsa) production, and in vitro evidence of transformation. As early as 10 days after infection, when more than half of the infected cells were gsa positive, FOCMA was detected on 5-10 percent of the cells. Transitory morphologic alterations (epithelioid appearance and rounding) were first noted in most cultures around 20-30 days post infection. At this time, approximately 50% of the cells in infected cultures expressed FOCMA. Morphologic characteristics of transformed fibroblastic cells (rounded shape, disordered alignment, and low adhesion to substratum), as well as enhanced agglutinability by plant lectins and ability to grow in agar, were demonstrated in one of four FeLV-infected, FOCMA-positive cultures. Findings showed that FOCMA may be expressed in FeLV-infected monolayer cells independent of transformation as assessed by in vitro criteria.

A micro-enzyme-linked immunosorbent assay (ELISA) test for detection of feline leukemia virus in cats

The performance of a micro ELISA test for detection of feline leukemia virus (FeLV) infection was evaluated. The test was found specific for FeLV and feline sarcoma virus (FeSV) group-specific antigens in blood, plasma or serum of infected cats. Other common feline pathogens were negative to the test. Quantities as little as 7.8 ng of p-27 (the major group specific antigen of FeLV) per ml of sample gave positive results. The correlation between the micro ELISA test and the indirect immunofluorescent test commonly used for diagnosis of FeLV infection was 98% in 116 clinical cases and 184 samples from cats inoculated with FeLV and 100% in 100 specific pathogen-free cats.

Rous sarcoma virus precursor protein pr 76 is processed in avian sarcoma virus-transformed mammalian cells after fusion-injection of viral protein p 15

Rat TWERC cells or hamster RBH cells which are transformed by avian sarcoma viruses synthesize small amounts of protein precursor pr 76 to the viral group-specific antigen (gag) but do not cleave it. Purified viral gag protein p 15 was trapped in resealed human erythrocyte ghosts and fusion-injected into the rat or hamster cells. Under these conditions the protein precursor pr 76 was intracellularly cleaved and one of the cleavage products, the viral gag protein p 27, was detected using an antiserum specific for protein p 27. These results suggest that the protein p 15 can be active under physiological conditions during the intracellular processing of protein precursor pr 76.

Localization of reverse-transcriptase in interferon-treated mouse cells chronically infected with Moloney leukemia virus.

Interferon treatment of mouse cells chronically infected with Moloney leukemia virus (3T3/MLV) resulted in 97 per cent inhibition of infective virus release. The intracellular localization and distribution of virus reverse-transcriptase and group specific (gs) antigen were determined in interferon treated and control cells. Cytoplasm of infected cells was fractionated by isopycnic centrifugation on discontinuous sucrose gradients. Fractions were analysed for their chemical composition and characterized by the activity of membranal marker enzymes. The association and levels of viral antigens were determined in each fraction. Fractions enriched with 5' nucleotidase, specific enzyme marker for plasma membrane, were also enriched with viral proteins. In interferon treated cells, intracellular accumulation of viral proteins was specifically localized in the plasma membrane. Threefold increase in reverse-transcriptase level was the maximal accumulation found in purified plasma membranes. Intracellular enzyme levels in interferon treated cells were in accordance with the amount of cell associated infective virus particles. The small accumulation of viral proteins and infective virus particles was not sufficient to account for the great reduction in virus yield observed in the supernatants of the interferon treated cells. A possible role for interferon in modification of plasma membrane associated with virus assembly is postulated.

Ev 3, a structural gene locus for endogenous virus, segregates with the gs +chf + phenotype in matings of line 6 3 chickens

ev 3 is one of seven recently described genetic loci of the chicken which contain structural genes for endogenous virus ( S. M. Astrin, 1978, Proc. Nat. Acad. Sci. USA 75, 5941–5945). ev 3 is present exclusively in chickens of the gs +chf + phenotype, that is, chickens whose cells produce endogenous viral group-specific antigen (gs) and viral envelope protein (chf). The segregation of ev 3 was followed in a genetic cross in which the gs +chf + phenotype was also segregating. The progeny of the cross were analyzed for endogenous viral loci by cleavage of embryo DNA with restriction endonuclease Sst 1, electrophoretic separation of the resulting fragments, and detection of bands containing viral sequences by hybridization of the DNA to radiolabeled viral RNA. Four endogenous viral loci, ev 1, ev 3, ev 4, and ev 5, were identified in the progeny of the cross. ev 1 was present in all of the progeny. ev 4 and ev 5 were present in progeny of both the gs +chf + and gs − chf − phenotypes and were observed to be genetically linked. ev 3 was present exclusively in progeny of the gs +chf + phenotype and all progeny of this phenotype contained ev 3. From these data, we suggest that ev 3 codes for gs and chf in the cells of gs +chf + chickens.

An Enzyme-Linked Immunosorbent Assay for Detecting Avian Leukosis-Sarcoma Viruses

Immunoglobulins from antiserum raised against chromatographically purified avian myeloblastosis virus (AMV) group-specific (gs) antigens were used in enzyme-linked immunosorbent assay (ELISA). Readily discernible color was produced with 2--3 ng of AMV protein in microplate wells coated with 4 micrograms of salt-precipitated immunoglobulins. When a biological assay, i.e., phenotypic mixing (PM), was the criterion for the infectious status of specimens, the ELISA consistently identified a greater percentage of virus-positive specimens than direct complement-fixation (DCF) tests. Over 95% concordance was obtained between the ELISA and PM bioassays when meconia and whole-blood samples were tested. Moreover, three DCF(-) egg albumens from one virus shedder hen were positive by the direct ELISA. Complete agreement was found between a biological assay for endogenous virus and the ELISA when blood and albumens from inbred chickens were tested. The ELISA is a rapid and convenient alternative to the DCF test for identifying infected chickens in eradication programs, because virus-rich sources such as meconia and blood that are unsuitable for DCF can be tested directly.

Immune response to avian leukosis virus infection in chickens: Sequential expression of serum immunoglobulins and viral antibodies

Chickens of a 15I 5 × 7 2 cross that produces endogenous Rous associated virus (RAV-0) were infected with subgroup A lymphoid leukosis virus (RAV-1). Within 3 weeks, before RAV-1 neutralizing antibodies were detected, significantly higher levels of serum immunoglobulin G (IgG) were found in infected birds than in uninoculated hatchmates. Immunoglobulin M was significantly elevated only during the late leukotic state. Although most of the inoculated birds tested had RAV-1 neutralizing antibodies, no correlation was found between IgG levels and antibody titers. Tolerance to endogenous virus (RAV-0) and viral group-specific antigen was apparently abrogated by RAV-1 inoculation because significantly higher percentages of iodinated envelope glycoprotein (gpE) of RAV-0 and a viral structural antigen of mol. wt 19,000 daltons (p 19) were precipitated by sera from inoculated birds than from control birds.

Endogenous viral genes of the White Leghorn chicken: common site of residence and sites associated with specific phenotypes of viral gene expression.

The DNAs from greater than 150 individual White Leghorn chickens were digested with restriction endonucleases BamHI, EcoRI, HindIII, and Sst I, fractionated by gel electrophoresis, denatured, and transferred to nitrocellulose filters. Fragments containing the endogenous viral genes were detected by hybridization with 70S Rous associated virus type 2[32P]RNA. Embryos of several different phenotypes with respect to production of the endogenous virus and expression of viral group-specific antigen and viral envelope protein were analyzed. DNA from birds of each phenotype produced a distinctive pattern of fragments containing viral genetic information. Individual fragments were seen to segregate as genetic loci in mating experiments. From the fragment patterns and the segregation data, the following conclusions were drawn with respect to the sites of residence in the chicken chromosome of the endogenous viral genes: (i) the DNA of all chickens contains viral genetic information in at least one site, and this site of residence appears to be the same in all chickens analyzed; (ii) four other sites have been identified, and the presence of viral information at each of these sites is always accompanied by a specific phenotype of endogenous viral gene expression; (iii) in addition to the above-mentioned five sites, a small number of other sites have been identified which are not associated with a known phenotype.

Further characterization of intracellular precursor polyproteins of rauscher leukemia virus

The identity and pathway of post-translational processing of viral primary gene products in cells infected with Rauscher leukemia virus were investigated in pulse-chase studies by precipitation with monospecific antisera and by the use of inhibitors of proteolytic cleavage. High molecular weight precursor polypeptides, designated Pr1 a+ b, of molecular weight of about 200,000, are precipitated both with antisera made against either one of the viral group-specific antigens (gag) p30, p15, p12, or p10 and with antisera made against the viral reverse transcriptase (RT). This indicates that these polypeptides contain determinants of both the “gag” and the polymerase (pol) gene products. Two pulse-labeled polypeptides, Pr3 (≅80,000–85,000 daltons) and its cleavage product Pr4 (≅70,000 daltons), are precipitated with anti-p30, -p15, -pl2, or -p10 sera, but not with anti-RT serum. Pr3 and Pr4 thus represent gag gene precursor polyproteins. Three other polypeptides, Pr RTl (≅145,000 daltons), Pr RT2 (≅135,000 daltons), and Pr RT3 (≅80,000–85,000 daltons), are precipitated with anti-RT serum only. Anti-gp69/71 serum precipitates the glycoprotein precursors Pr2 a+ b (≅90,000 daltons), but none of the above-mentioned polypeptides. Quantitative measurements of pulse-chase profiles and results obtained with the inhibitor TPCK and the arginine analog canavanine indicate that the proteolytic cleavage of Pr1 a+ b is the primary pathway for the formation of intermediate RT precursors Pr RT1, 2, 3. The same measurements did not clarify whether or not the cleavage of Pr1 a+ b is involved in the formation of gag gene products. However, they indicated that most of the detected amount of the gag gene precursors Pr3 and Pr4 is made by a mechanism that does not involve the cleavage of preformed Pr1 a+ b. A possible explanation of these results is suggested in a model in which translation proceeds from one initiation site to give rise either to a gag gene product or, less frequently, to a common gag-pol precursor that is made by overriding a termination site at the end of the gag gene.

Preparation of Antisera to Group-Specific Antigens of Avian Leukosis-Sarcoma Viruses: An Alternate Approach

Immunization of rabbits with a pooled preparation of chromato-graphically purified avian myeloblastosis virus (AMV) group-specific (gs) antigens produced relatively large volumes of antiserum that was as broadly reactive in complement-fixation (CF) tests as antiserum produced in hamsters with tumors induced by Rous sarcoma virus. This alternate procedure should be of value for routine preparation of leukosis virus gs antiserum. Other antisera prepared against disrupted AMV had spurious reactivities that confused CF testing of embryos and tissue extracts. This artifact was attributed to the high affinity of serum albumin for AMV virions and the resultant production of antibodies when plasma-derived AMV was used as immunogen. A high-molecular-weight AMV CF antigen was also found in early fractions eluted from an agarose-gel column.

Biosynthesis of subviral oncogenic particles (virosomes) in mitochondria of rous sarcoma and Rauscher murine leukemia cells

Experimental evidence for the presence and biosynthesis of subviral, leukemogenic particles in the isolated mitochondria of spleen cells of mice infected with Rauscher murine leukemia (RML) virus is presented. These subviral particles sediment at a density of 1.27-1.29 g/ml and induce splenomegaly and RML three weeks after i.v. or i.p. administration to white mice. Virosomes have been labelled with [32P]phosphate in the isolated mitochondria from RML spleen cells and high molecular weight (70S) [32P]RNA has been isolated from these subviral, leukemogenic particles. Rauscher virus group specific antigens were detected by immunodiffusion in the inner membrane and matrix fraction of the mitochondria of RML spleen cells. These results together with our earlier findings strongly suggest that mitochondria of the transformed cells participate in the biosynthesis of RNA tumor viruses. Possible mechanism of the penetration of viral genetic information of RNA tumor viruses into mitochondria of tumor cells in vivo is discussed.

Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses

Nondefective and transformation-defective virion subunit RNAs from two strains of Rous sarcoma virus (RSV) were translated in cell-free systems derived from Krebs IIA ascites cells, wheat germ, and L-cells. In each case the predominant viral-specific product was a polypeptide of molecular weight 76,000 that is related to the internal viral group-specific antigens, as judged by immunoprecipitation with monospecific antisera and tryptic peptide fingerprinting. No difference could be detected between the translation products of 35S RNA from nondefective and transformation-defective RSV virions, nor of 35S RNA from different strains of RSV. The 76,000-molecular-weight polypeptide synthesized in response to 35S RNA in vitro was labeled with formyl-methionine from initiator tRNA. Models for viral protein synthesis are discussed in the light of these results, and arguments positioning the group-specific antigen gene at the 5' end of the 35S RNA are presented.

Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. III. Alterations of splenic B and T cells in Friend virus-infected mice.

Lymphoid tissues of mice infected with murine leukemia virus (Friend) (MuLV-F) were examined for the presence of cellular markers of MuLV-F infection. The Friend virus-associated cell membrane antigen (FVMA) and the virus group-specific antigen (GSA) were detectable on cells from the spleen and, to a lesser degree, on cells from the bone-marrow. In contrast, neither FVMA nor GSA was found in cells from the thymus. Alterations in the B-cell and T-cell spleen populations of MuLV-F-infected mice were then studied. The proportion of Ig-positive cells declined from the initial 45% (in non-infected controls) to about 10% after 2 weeks of infection. A similar decline of theta-positive cells was noted. However, complement-bearing cells (EAC rosettes) declined even more rapidly and became undetectable in the second week after infection. The treatment of spleen cells from MuLV-F-infected mice with anti-FVMA serum plus complement in vitro reduced the number of detectable Ig-positive cells, specifically, whereas the number of theta-positive cells remained unchanged. Furthermore, B and T cells from spleens of infected mice were separated on an affinity column with anti-Ig antibody-coated beads. The initial cell suspension contained about 45% FVMA-positive cells, about 40% Ig-positive cells and about 40% theta-positive cells. Ig+ cells were retained on the column. The theta-positive cell fraction was collected in the eluate and contained very few FVMA-positive cells with some "null" cells. Most of the FVMA-positive cells were retained on the column, which strongly suggested that they were B cells. These results confirm the previous experiments which showed the selective infections of purified splenic B cells by MuLV-F in cultures.

Endogenous ecotropic mouse type C viruses deficient in replication and production of XC plaques.

Endogenous ecotropic type C viruses were induced by iodedeoxyuridine from nontransformed and chemically or spontaneously transformed clones of the C3H/10T1/2 cell line. Viruses produced by cells of certain transformed clones were N-tropic and formed large XC plaques. In contrast, viruses produced by nontransformed C3H/10T1/2 cells were not detectable in the XC plaque test. These XC- viruses infected mouse cells with high efficiency, as shown by the induction of murine leukemia virus group-specific antigens in infected cells, but virus production, as determined by DNA polymerase-containing particles, was extremely low. Upon growth in certain mouse cells these replication-deficient, XC(-) viruses converted to type C viruses that were similar in XC assays to N-tropic AKR virus (XC+).

Lack of correlation between Marek's disease tumor induction and expression of endogenous avian RNA tumor virus genome

Infection with Marek's disease virus (MDV) and subsequent tumor development failed to induce the expression of endogenous avian RNA tumor virus genome in Line-15I and Line-7 chickens which lacked such expression at 1 day of age. Titers of avian leukosis virus (ALV) group-specific (gs) antigen detected by the COFAL test and expression of chick helper factor (chf) activity remained relatively constant in birds which expressed these genome functions at hatching time. Endogenous ALV belonging to the E-subgroup was isolated from two of 50 birds; both were controls not exposed to MDV. There was no correlation between expression of ALV genome (gs or chf) at 1 day of age and the rate of tumor development subsequent ot MDV infection. It was concluded that MDV infection can induce tumors without the participation of endogenous ALV genome.

Genetic control of type C virus of wild mice

THE genetic control of type C virus group-specific (GS) antigen expression1,2, infectious virus3–6, and tumorigenesis6,7 has been described in crosses between strains of inbred laboratory mice with high and low incidence of leukaemia. We described a natural population of wild mice (Mus musculus) near Lake Casitas (LC) in southern California which showed a high level of indigenous type C virus activity in terms of GS antigen and infectious virus throughout their lifetime. When ageing was observed in the laboratory, LC wild mice showed a marked susceptibility to lymphoma8, epithelial tumours9 and a neurogenic hind leg paralysis10. Virus transmission and in vivo neutralisation studies11 established the indigenous type C virus as the critical determinant of the lymphoma and paralysis. To ultimately prevent these diseases by antiviral measures we have attempted to suppress LC virus expression by crossbreeding LC wild mice with a specific inbred laboratory mouse strain C57BL/10 Snell (designated B10). The B10 mouse was selected because of its homozygosity for two non-linked dominant alleles, Fv-1b and MLv-1b, the function of either of which might be expected to restrict virus expression in the hybrid progeny. Laboratory strains of mouse type C virus can be classified according to their in vitro host range as N-, B-, or X(xenotropic)-tropic depending on whether they grow preferentially in NIH Swiss or BALB/c embryo cells or replicate only in non-murine cells12,13. Field isolates from LC mice have an unusually wide in vitro host range (‘amphotropic’) but are N-tropic for mouse cells (S. Rasheed, V.K., and M.B.G., unpublished). B10 mice, within the first year of life, show a low incidence of both N-and B-tropic infectious virus14 and also harbour an endogenous X-tropic genome13. The Fv-1b allele from the B10 parent should limit the replication of N-tropic virus within the LC×B10 progeny15, presumably by interfering with the integration or transcription of the proviral DNA16. The MLv 1b allele, the function of which is unknown, might restrict either N-, B-, or X-tropic virus expression in the progeny since this allele completely suppressed GS antigen expression in B10 crosses with the congenic resistant (58N) strain17. Although the genetic mechanism has yet to be established, our findings indicate that control of type C virus expression can be accomplished by genetic means in wild mice.

Feline Oncornavirus-Associated Cell Membrane Antigen. III. Antibody Titers in Cats From Leukemia Cluster Households<xref ref-type="fn" rid="fn2">2</xref>

Serum samples from 182 healthy cats residing in environments known to have a natural exposure to feline leukemia virus (FeLV) were examined for the presence of antibody to the feline oncornavirus-associated cell membrane antigen. The number included 138 cats from leukemia-"cluster" households. Such cats previously showed greatly increased frequencies of FeLV infection, as determined by the virus group-specific antigen (gasa) in peripheral blood leukocytes and platelets. The geometric mean antibody titer for all 182 cats with known exposure to FeLV was 4.69, more than four times higher than the mean titer for healthy pet cats from the same geographic areas but with no known FeLV exposure. About 92 percent of the cats in exposure environments were positive for FeLV gsa or feline oncornavirus-associated cell membrane antigen (FOCMA) antibody, and gsa-positive cats had lower FOCMA antibody geometric mean titers than gsa-negative cats. In the exposure environments, no differences were seen for cats of different sexes, but a higher geometric mean antibody titer was observed for cats 5 years and over when compared to younger groups. These results prove that previous reports of increased incidences of leukemia in cluster households were not due to chance alone, but rather to increased infection rates of cats in the FeLV environment. Also, they suggest that horizontal transmission of FeLV is highly efficient under crowded conditions, and that most cats naturally exposed to virus elicit an active immune response.