Mutants of nonproducer cell lines transformed by murine sarcoma virus. 3. Detection and characterization of RNA specific for helper and sarcoma viruses.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Immune virolysis: effect of antibody and complement on C-type RNA virus.

In the presence of envelope antibody and complement, the AKR strain of mouse leukemia virus was lysed, with the result that (i) the viral nucleic acid became susceptible to ribonuclease digestion and (ii) the internal group-specific antigen of the virus was released. The internal localization of the group-specific antigen is confirmed, the evidence being based on the failure of group-specific antibody to lyse virus in the presence of complement.

Amino acid sequence homology between histone H5 and murine leukemia virus phosphoprotein p12.

The amino terminal acid sequences of several mouse leukemia virus phosphoproteins (p12) show definite homology with the amino terminal conserved region of H5 histones, the phosphorylated nuclear proteins of nucleated erythrocytes. Differences in the amino acid compositions of the two groups of proteins seem to rule out the possibility that they evolved from a single common ancestral gene. The finding of sequence homology between viral p12's and cellular histones, however, is consistent with evolution of retrovirus structural proteins by a process of differentiation from preexisting cellular genes. The conserved primary and secondary structure at the amino terminal region, common to both groups of proteins, may be related to their common function of nucleic acid binding modulated by phosphorylation.

Interactions of immunoglobulins G and M in the detection of the mammalian C-type virus cross-reactive antigen.

The mammalian C-type tumor viruses share an antigenic determinant, gs-3, located on the major internal polypeptide of the virion. Detection of this determined in gel diffusion assays by antiserums prepared in rats by immunization with rat tumor homogenates carrying murine virus and serums prepared in a rabbit by immunization with purified murine gs antigen depended on antibodies present in the fractions containing immunoglobulins M and G. The immunoglobulin G fraction by itself precipitated only the homologous murine antigen. Neither fraction alone precipitated heterologous (cat, rat, or hamster) antigen (definition of the gs-3 reaction), while a mixture of the two fractions did. The gs-3 reaction was eliminated by treatment of the serums with beta-mercaptoethanol, also indicating a requirement for immunoglobulin M antibodies.

Feline leukemia and RD-114 virus group-specific proteins: comparison of amino terminal sequence.

The major internal virion polypeptide from feline and RD-114 type C viruses has been subjected to amino terminal sequence analyses with the Beckman automated sequencer. These proteins, as well as their homologs in rat and mouse viruses, begin with the sequence prolylleucylarginyl (Pro-Leu-Arg). Virus RD-114 differs from conventional feline type C viruses that show about 80 percent relatedness based on calculation of the minimum number of base changes to give equivalent coding for the protein segments analyzed. In addition, insertion of a gap in the RD-114 sequence is necessary to maintain positional homology. The difference between RD-114 and feline leukemia virus appears as great as the difference between mouse type C viruses and either of these two viruses. Thus, even though current evidence suggests that RD-114 is of feline origin, the sequence differences between RD-114 and conventional feline virus group-specific proteins is well beyond that based on one or a few point mutations.

Isolation and characterization of an endogenous type C virus of rhesus monkeys.

A type C retrovirus was isolated from a continuous cell line established from a spontaneous esophageal carcinoma of a rhesus monkey (Macaca mulata) by prolonged cocultivation with canine cells. A DNA transcript of the viral RNA hybridized to a high level and kinetic analysis indicated the presence of multiple copies of the viral genome in rhesus monkey DNA, showing that the virus is endogenous in this species. The rhesus monkey virus closely resembles, in several respects, an endogenous type C virus previously isolated from stumptailed macques (Macaca arctoides), aa species closely related to rhesus monkeys.

Feline leukemia and sarcoma viruses: susceptibility of human cells to infection.

Human embryonic cells are highly susceptible to infection with feline leukemia and sarcoma viruses. These viruses were propagated in human cultures without antigenic modification or loss of infectivity for cat or human cells. Virus stocks contained at least 10(6) infectious units of virus per milliliter for human cells. Virus present in 10(-6) dilution of virus stock replicated to detectable amounts as early as 7 days after virus infection. The feline sarcoma virus induced morphological transformation of human cells.

Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS.

The two main subgroups of the family of human T-lymphotropic retroviruses (HTLV) that have previously been characterized are known as HTLV-I and HTLV-II. Both are associated with certain human leukemias and lymphomas. Cell surface antigens (p61 and p65) encoded by HTLV-I are frequently recognized, at low titers, by antibodies in the serum of patients with acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that precede AIDS (pre-AIDS). This suggests an involvement of HTLV in these disorders. Another subgroup of HTLV, designated HTLV-III, has now been isolated from many patients with AIDS and pre-AIDS. In the studies described in this report, virus-associated antigens in T-cell clones permanently producing HTLV-III were subjected to biochemical and immunological analyses. Antigens of HTLV-III, specifically detected by antibodies in serum from AIDS or pre-AIDS patients and revealed by the Western blot technique, are similar in size to those found in other subgroups of HTLV. They include at least three serologically unrelated antigenic groups, one of which is associated with group-specific antigens (p55 and P24) and another with envelope-related (p65) proteins, while the antigens in the third group are of unknown affiliation. The data show that HTLV-III is clearly distinguishable from HTLV-I and HTLV-II but is also significantly related to both viruses. HTLV-III is thus a true member of the HTLV family.

Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus.

A study was conducted of the genetic relation between human T-cell lymphotropic retroviruses and visna virus. The human T-cell lymphotropic viruses include those associated with T-cell malignancies (HTLV-I and HTLV-II) as well as the etiologic agent of the acquired immune deficiency syndrome (HTLV-III). Visna virus, a slowly replicating and pathogenic but nononcogenic retrovirus of sheep, is a member of the subfamily Lentivirinae. Results obtained by molecular hybridization and heteroduplex analysis indicated that a greater extent of nucleotide sequence homology exists between HTLV-III and visna virus than between HTLV-III and any of the other viruses. The homology observed under conditions of low stringency spanned the entire genome, but was strongest in the gag/pol region. The morphogenesis and fine structure of HTLV-III and visna virus also demonstrated striking similarities. The data provide strong evidence for a close taxonomic and thus evolutionary relation between HTLV-III and the Lentivirinae subfamily.

Simian AIDS: isolation of a type D retrovirus and transmission of the disease.

A type D retrovirus related to but distinct from Mason-Pfizer monkey virus was isolated in vitro from the blood of two rhesus monkeys (Macaca mulatta) with simian acquired immunodeficiency syndrome (SAIDS). Three juvenile rhesus monkeys that were injected intravenously with tissue culture fluids containing this virus developed SAIDS after 2 to 4 weeks.

Humoral immune responses of cats to feline leukemia virus: comparison of responses to the major structural protein p30 and to a virus-specific cell membrane antigen (FOCMA).

Radioimmunoprecipitation was used to test cat sera for ability to bind to the purified major internal protein p30 of feline leukemia viurs (FeLV), to the endogenous cat virus (RD-114), and to murine leukemia virus (MuLV). The data were compared with results of tests for antibody to the feline oncornavirus-associated cell membrane antigen FOCMA and for the presence of viremia. In contrast to the general lack of free antibody to FeLV p30 in a random sample of healthy cats, high levels of antibody to FeLV p30 and FOCMA were found in normal animals from high-leukemia-cluster households. Titers of greater than or equal to 200 for p30 and greater than or equal to 32 for FOCMA were found in nonviremic animals; a percentage of animals with high FOCMA titers and lower or no p30 binding activity were viremic. Animals with neoplasms were low or negative for FOCMA antibody and did not have high titers of free p30 antibody. The p30 binding activity could be divided into three main categories: high binding with FeLV p30 and much lower activity with RD-114 and MuLV p30's, as seen with hyperimmune sera; high binding with FeLV and RD-114 p30's and low activity with MuLV p30, possibly indicative of specific antibody to both of the aforementioned proteins; and low level binding to all three p30's.

Natural and experimentally induced antibodies to defined mammalian type-C virus proteins in primates.

Using sensitive radiommunoprecipitation assays for highly purified type-C RNA tumor virus proteins, we found that 5 of 16 clinically normal gibbons (including 4 of 5 normal animals from a colony with 2 cases of lymphoma) and 4 of 4 experimentally inoculated gibbons formed antibodies to the major structural protein (p30) of gibbon ape leukemia virus (GaLV). An additional woolly monkey immunized with the closely related simian sarcoma virus also formed antibodies detectable with GaLV p30. Of 20 patients immunized with formalin-inactivated Rauscher murine leukemia virus (R-MuLV), 10 were previously reported to have antibodies to MuLV as determined by an internally labeled banded virus radioimmunoprecipitation assay. In comparison studies with purified R-MuLV proteins, 7 of 20 patients formed antibodies: 3/20 to R-MuLV p30 only, 1/20 to R-MuLV glycoprotein (gp) 70 only, and 3/20 to both p30 and gp70. Most responders were melanoma patients receiving immunotherapy with BCG. Additionally, rhesus monkeys produced antibodies to the endogenous cat virus RD114 and closely related endogenous baboon leukemia virus p30's. Thus these studies demonstrated the ability of primates (including humans) to form antibodies to well-characterized proteins from endogenous and exogenous type-C viruses and the potential utility of these assays for seroepidemiologic studies.

Suppression of infectious murine leukemia virus in wild mice (Mus musculus) by passive immunization.

Passive immunization with heterologous antivirus antiserum beginning at birth successfully suppressed infectious murine leukemia virus expression in Lake Casitas wild mice (Musmusculus) at 5-7 weeks of age.

Prevention of spontaneous leukemia in AKR mice by passive immunization and type specificity of this protection.

AKR mice were given sc injections of goat antiviral immune globulin from birth through 14 or 31 days of age. Although the shorter immunization schedule greatly reduced expression of spontaneous leukemia, the longer immunization period was required for essentially complete prevention of leukemia. Immune globulin with a high neutralization titer for ecotropic virus provided this protection, whereas antibody with a high neutralization titer for murine xenotropic virus did not.

Inefficient humoral immune response of lymphoma-prone wild mice to persistent leukemia virus infection.

Adult wild mice (LC) from a natural colony with a high incidence of spontaneous lymphomas had free infectious virus in their seara (average 10(3.5) infectious units/ml) and parenchymal organs (average 10(5.2) infectious units/g tissue). They did not have detectable levels of free virus-specific antibodies that could be demonstrated by virus neutralization or immunofluorescence at higher than a 1:10 dilution. Only 5 of 28 animals had free antibodies detectable by radioimmunoprecipitation assay, and tissues of 4 mice also had nondetectable levels of virus determined by infectivity assay. Formalized vaccine from the indigenous virus did not induce production of virus-neutralizing antibodies or protect against naturally occurring disease. The animals with persistent leukemia virus infection, however, elicited good humoral immune responses to virus-unrelated antigen.

Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species.

BACKGROUND: In the autumn of 1992, a novel form of chronic, active hepatitis of unknown etiology was discovered in mice at the National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, Md. A high incidence of hepatocellular tumors occurred in affected animals. The disease entity was originally identified in A/JCr mice that were untreated controls in a long-term toxicologic study. PURPOSE: Our original purpose was to determine the origin and etiology of the chronic hepatitis and to quantify its association with hepatocellular tumors in mice of low liver tumor incidence strains. After a helical microorganism was discovered in hepatic parenchyma of diseased mice, we undertook characterization of the organism and investigation of its relationship to the disease process. METHODS: Hepatic histopathology of many strains of mice and rats, as well as guinea pigs and Syrian hamsters, in our research and animal production facilities was reviewed. Steiner's modification of the Warthin-Starry stain and transmission electron microscopy were used to identify bacteria in the liver. We transmitted the hepatitis with liver suspensions from affected mice and by inoculation with bacterial cultures. Bacteria were cultivated on blood agar plates maintained under anaerobic or microaerophilic conditions and characterized morphologically, biochemically, and by 16S rRNA sequence. RESULTS: We report here the isolation of a new species of Helicobacter (provisionally designated Helicobacter hepaticus sp. nov.) that selectively and persistently colonizes the hepatic bile canaliculi of mice (and possibly the intrahepatic biliary system and large bowel), causing a morphologically distinctive pattern of chronic, active hepatitis and associated with a high incidence of hepatocellular neoplasms in infected animals. CONCLUSIONS: The novel Helicobacter is a likely candidate for the etiology of hepatocellular tumors in our mice. The Helicobacter-associated chronic active hepatitis represents a new model to study mechanisms of carcinogenesis by this genus of bacteria. IMPLICATIONS: Adenocarcinoma of the stomach, the second most prevalent of all human malignancies world-wide, is associated with infection at an early age with Helicobacter pylori. Infection leads to several distinctive forms of gastritis, including chronic atrophic gastritis, which is a precursor of adenocarcinoma. H. hepaticus infection in mice constitutes the only other parallel association between a persistent bacterial infection and tumor development known to exist naturally. Study of the H. hepaticus syndrome of chronic active hepatitis and liver tumors in mice may yield insights into the role of H. pylori in human stomach cancer and gastric lymphoma.

Effects of hydroxyurea on murine type C virus-specific DNA synthesis in newly infected cells.

Cell transformation and replication of the Rauscher pseudotype of Moloney murine sarcoma virus in mouse embryo fibroblasts were inhibited by hydroxyurea within a critical time period of 30 to 90 min postinfection. In cells infected by Rauscher leukemia virus, treatment with 1mM hydroxyurea during the critical time period resulted in the accumulation of minus-strand DNA (molecular weight, 3 x 10(6)) in association with the parental viral genoma RNA. This 5 to 6 x 10(6) dalton RNA:DNA hybrid was found in the cytoplasm. Positive-strand DNA of genomic or smaller size was not detected in the presence of hydroxyurea, but virus-specific DNA was found in the nucleus 30 min after removal of drug.

Heteroduplex mapping in the molecular analysis of the human T-cell leukemia (lymphotropic) viruses.

The human T-cell lymphotropic virus (HTLV) family includes members associated with T-cell cancers (HTLV-I and HTLV-II) as well as the etiological agent of the acquired immunodeficiency syndrome (HTLV-III). Molecular clones of these viruses were used in heteroduplex mapping experiments to study their structural and evolutionary relationships. The HTLV-I subgroup, despite some restriction enzyme site polymorphism, demonstrated a high degree of sequence conservation. Heteroduplexes of HTLV-I and HTLV-II demonstrated a significant amount of sequence homology, with the strongest region of conservation occurring in the 3'-most coding sequences, designated pX, and to a lesser, although substantial extent in the rest of the genome. Thus, the genomic organization of HTLV-II appears to be very similar to that of HTLV-I. All HTLV-III molecular clones appeared to be identical, with a single exception, which showed heterogeneity in the env gene region. In heteroduplexes between HTLV-I and HTLV-III, very little homology was observed, being confined to the gap/pol region. In contrast to the latter result, a striking amount of homology was detected between HTLV-III and a morphologically similar, pathogenic, nononcogenic lentivirus, visna virus. These data provide strong evidence for a close taxonomic and thus evolutionary relationship between HTLV-III and the lentivirus subfamily of retroviruses. A taxonomic tree, based on the genetic relatedness and biological properties of the HTLV family, is proposed.

An unlabeled antibody macromolecule technique using hemocyanin for the identification of type B and type C retrovirus envelope and cell surface antigens by correlative fluorescence, transmission electron, and scanning electron microscopy.

The present resolution (75-100 A) of the conventional scanning electron microscope (SEM) and its ability to image the surfaces of large numbers of whole cells in situ permit the approach of problems such as viral and cell surface antigen localization by immunological labeling with visual markers. Identification of virus and cell surface antigens in situ has been accomplished in indirect reactions by unconjugated markers. Hemocyanin (Hcy) from whelk, Busycon canniculatum, has been developed as an immunospecific marker for virion and cell surface labeling in the electron microscope. Its size (30 x 50 nm) and distinct cylindrical shape permit easy visualization in the SEM and the transmission electron microscope (TEM). The Hcy method involves the preparation of antisera to Hcy in appropriate hosts for use in an unlabeled antibody macromolecule procedure based exclusively on antigen-antibody affinity to couple the macromolecule to the antigen site. Further correlative data from fluorescence microscopy can be obtained from similarly labeled samples by binding fluorescein to the bridging antibodies used in the Hcy technique. The usefulness of the Hcy marker system was demonstrated by employing highly specific antisera to the major envelope and cell surface glycoprotein (gp70) of Rauscher murine leukemia virus (R-MuLV), a type C retrovirus. The antiserum was shown to bind to the virion and cell surfaces of virus-infected cells in the homologous virus-infected cell system. It also demonstrated the expression of R-MuLV gp70-related antigens on a murine cell line Mm5mt/c1 which produces mouse mammary tumor virus (MuMTV), a type B retrovirus. Furthermore, when used in the Hcy marker system the anti-gp70 serum was able to distinguish type B from type C budding virus on the same cell. Methods for the preparation of immunoreagents and labeling of cells are discussed.

Microinjection and expression of an infectious proviral clone and subgenomic envelope construct of a human immunodeficiency virus.

An infectious proviral clone of the human immunodeficiency virus (HIV) was microinjected into the cell nucleus in six cell lines derived from caprine, ovine, bovine, or human solid tissue to study the utility of this method in effecting viral gene expression in nonlymphoid cells. Immunofluorescence assays for HIV demonstrated viral gene expression in only 5% of cells (100-200 cells per line) 24-48 h after microinjection; however, no reverse transcriptase activity was detectable, presumably due to a low level of virus release in this limited number of cells. Therefore, to indirectly assess infectious virus release, microinjected cells were cocultured with human T4 antigen-positive lymphocytes (H9) sensitive to HIV infection. Syncytia formation, electron microscopy, reverse transcriptase activity, and radioimmunoassay for HIV p24 were used to monitor viral gene expression in cocultures. HIV was efficiently recovered by cocultivating H9 with microinjected cells 48 h after microinjection, regardless of the tissue type or species of origin. H9 syncytia were visualized in some cocultures as early as day 5 but were readily apparent in all experiments on days 7-10. Syncytia induction in H9 was the earliest and most reliable indicator of infectious virus release. A recombinant construct containing a subgenomic envelope gene derived from the proviral clone of HIV was microinjected into human glioblastoma cells. Twenty-four to 48 h after manipulation, 5-20% of microinjected cells were found by immunofluorescence assay to express low levels of a putative gp120. These results suggest a possible approach to producing virus-free HIV envelope antigens in mammalian cells and may be relevant to subunit vaccine development.