Mouse mammary tumor virus in hybrids from strains C57BL and GR: breeding test of backcross segregants.

F1 and F2 and first backcross hybrids and second backcross families of the high mammary tumor incidence strain GR and the low incidence strain C57BL were examined for the segregation of mouse mammary tumor viral (MMTV) expression. Although GR has been reported to transmit MMTV as a single dominant gene, several lines of evidence suggest there are multiple genetic factors that influence MMTV expression, MMTV expression as measured by double antibody radioimmunoassay for MMTV p14 segregated in 106 first backcross progeny at a 60:40 ratio, intermediate between what would be expected for either a single or two gene hypothesis. In female second backcross progeny of either male or female first backcross, a heterogeneous pattern of expression was noted that does not fit any simple Mendalian pattern. From an analysis of serial lactations of first backcross and second backcross families, it appears that all hybrid females contain MMTV proviral information that may be expressed either at late lactations or in a variable proportion of progeny mice. These combined results are most consistent with a vertically transmitted genome regulated by multiple factors in these crosses.

Mammary tumors and mammary tumor virus expression in hybrid mice of strains C57BL and GR.

Mammary tumorigenesis in genetic crosses between the high mammary tumor incidence GR and the low incidence C57BL mouse strains is highly correlated with murine mammary tumor virus expression in milk. Although the F1 and first backcross females had a mammary tumor incidence which was consistent with a single dominant gene segregation, the tumor incidence in the critical second backcross segregants disproved the single gene hypothesis. Genetic factors were clearly involved in regulation of virus expression which in turn correlated with both tumor incidence and tumor latency; these complex phenotypes are however best explained as threshold or quasicontinuous characters. As predicted from this model, the age specific incidence of mammary tumors showed a broad peak at 14-19 mo of age with no evidence of an early or late phase. Hematopoietic tumors showed no correlation with virus expression or mammary tumorigenesis suggesting different etiologies for these tumors.

Adeno-associated satellite virus interference with the replication of its helper adenovirus.

Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80 degrees C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This suggested that all cells infected with the two viruses were producing a reduced quantity of adenovirus.

Radioimmunoassay of mammalian type C viral proteins. 3. Detection of viral antigen in normal murine cells and tissues.

A radioimmunoassay specific for a murine leukemia virus structural protein, the gs antigen, detects an antigenic reactivity in normal murine cells in culture and natural tissues. The assay was shown to measure an antigen that is highly related to the virion protein as shown by absorption tests, immunoadsorbent chromatography, and by analysis of linearized dose-response curves. These findings combined with the finding of viral-specific RNA indicate that portions of the viral genome are being expressed with a much greater frequency than previously appreciated.

Dexamethasone stimulation of murine mammary tumor virus expression: a tissue culture source of virus.

In mouse cell lines derived from mammary adenocarcinomnas, the synthetic steroid dexamnethasone stimulates production of murine mammary tumor virus. Viral RNA and antigens are increased as much as 20-fold, and culture fluid supernatants from steroid-treated cells contain type B particles with reverse transcriptase. These cells provide a possible tissue culture source of this virus and a model system for studying the mechanism of action of corticosteroids and the regulation of transcription of integrated viral DNA.

Reverse transcriptases of primate viruses as immunological markers.

Antibodies were prepared against the DNA polymerases (reverse transcriptases) of three potentially oncogenic RNA viruses of primates. Two type C viruses, isolated from a woolly monkey fibrosarcoma and from a gibbon ape lymphosarcoma, have polymerases that are immunologically related to each other and are distinct from the type C viruses isolated from other mammals.

Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS.

In a study of genetic variation in the AIDS virus, HTLV-III/LAV, sequential virus isolates from persistently infected individuals were examined by Southern blot genomic analysis, molecular cloning, and nucleotide sequencing. Four to six virus isolates were obtained from each of three individuals over a 1-year or 2-year period. Changes were detected throughout the viral genomes and consisted of isolated and clustered nucleotide point mutations as well as short deletions or insertions. Results from genomic restriction mapping and nucleotide sequence comparisons indicated that viruses isolated sequentially had evolved in parallel from a common progenitor virus. The rate of evolution of HTLV-III/LAV was estimated to be at least 10(-3) nucleotide substitutions per site per year for the env gene and 10(-4) for the gag gene, values a millionfold greater than for most DNA genomes. Despite this relatively rapid rate of sequence divergence, virus isolates from any one patient were all much more related to each other than to viruses from other individuals. In view of the substantial heterogeneity among most independent HTLV-III/LAV isolates, the repeated isolation from a given individual of only highly related viruses raises the possibility that some type of interference mechanism may prevent simultaneous infection by more than one major genotypic form of the virus.

Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants.

Multiple human immunodeficiency virus type-1 sequences from the V3 and V4-V5 regions of the envelope gene were analyzed from three mother-infant pairs. The infants' viral sequences were less diverse than those of their mothers. In two pairs, a proviral form infrequently found in the mother predominated in her infant. A conserved N-linked glycosylation site within the V3 region, present in each mother's sequence set, was absent in all of the infants' sequence sets. These findings demonstrate that a minor subset of maternal virus is transmitted to the infant.

Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic "hot spot" structure, a subcellular distribution that is characteristic of Tax in HEK cells.

Antigenic determinants in a virus-induced mouse mammary tumor recognized by cell-mediated immune assays.

Purified tumor cell membrane (PTCM) fractions from spontaneous BALB/cfC3H mammary adenocarcinomas elicit blastogenic responses in spleen cells of tumor-bearing mice. Previous studies have demonstrated that splenic T-cells are the major responding cell population but have not clarified whether the antigens responsible for the reactions are exclusively viral in origin or may involve nonviral tumor-associated antigens (TAA). Pretreatment of PTCM preparations with polyvalent anti-murine mammary tumor virus (MuMTV) completely obliterated the ability of PTCM to stimulate an innocent bystander cytotoxicity reaction; however, it reduced the blastogenic response by only 60%, suggesting that the two assays may measure different antigenic reactivities. The specificity of the anti-MuMTV blocking for viral antigens was demonstrated by the complete absorption of the serum-blocking reaction with purified MuMTV particles in the cytotoxicity assay; however, absorption was only partial in the blastogenesis assay. Incubation with purified Rauscher murine leukemia virus particles failed to absorb the neutralizing effect of the sera. These data suggest that putative TAA can be detected in the blastogenesis assay but not in the cytotoxicity reaction. Ammonium sulfate-precipitated immunoglobulin fractions of sera from tumor-bearing BALB/cfC3H mice (TBMS) completely blocked the stimulatory potential of PTCM in both the blastogenesis and the cytotoxicity assays. In the reciprocal experiment to that described above, preabsorption of TBMS immunoglobulins with purified MuMTV completely removed the inhibition of the cytotoxicity but again only partially restored the blastogenic response. The reaction could be completely restored by preabsorption of TBMS with PTCM. These results support the contention of nonvirion antigen involvement in the blastogenesis reaction. In conclusion, these two assay systems detect different antigenic determinants on the MuMTV-expressing tumor cell membranes. Both viral and other antigens appear to be relevant in this model system, and serum factors present in the immunoglobulin fractions of tumor-bearing mice can inhibit T-cell responses directed at either kind of antigenic moieties.

Dependence of initiation of an innocent bystander cytotoxicity reaction on the association of viral antigens and tumor cell membranes in mice.

In chemically induced mouse mammary tumors in BALB/c mice, no murine mammary tumor virus (MuMTV) antigens could be detected in the tumor cell membranes. In contrast, in mammary tumors of spontaneous appearance in BALB/cfC3H mice neonatally infected with MuMTV, viral antigens could be detected by immunofluorescence. Specific activation of immune spleen lymphocytes in vitro by homologous tumor cell membrane preparations resulted in an innocent bystander cytotoxicity reaction in BALB/c mice bearing either the chemical- or virus-induced mammary tumors. Non-tumor bearers did not respond in this reaction. The cytotoxicity effector cell was a nylon-adherent Thy 1.2+, Lyt 1+, Lyt 2+ lymphocyte. Induction of the reaction in the chemically induced mammary tumor bearers was related to tumor-associated antigens, but in the mice with MuMTV-induced tumors, it was possible that all responses were solely due to viral antigens. Biochemical analyses using immunoprecipitation techniques indicated that the major external glycoprotein of MuMTV (gp52) was present in the tumor cell membrane preparations. Preincubation of the cell membranes of virus-induced tumors with anti-MuMTV completely blocked the capacity of this preparation to induce the cytotoxicity. Preabsorption of the anti-MuMTV with purified MuMTV removed all the blocking activity of these sera, indicating that the antigenic determinants recognized were of viral origin. However, purified MuMTV failed by itself to elicit the innocent bystander cytotoxicity. This observation indicates that an association with membranes was necessary for the viral antigens to initiate and/or effect this cell-mediated immune reaction.

Lymphoproliferative responses to mouse mammary tumor virus in lymphocyte subsets of breast cancer patients.

Altered immunologic reactions were observed in breast cancer patients as compared to those in normal subjects. Lymphoproliferative responses to murine mammary tumor virus (MuMTV) were significantly enhanced in peripheral blood mononuclear cells from patients with metastatic disease. These reactivities occurred with mammary tumor virus purified from either mouse milk or infected feline kidney cells but not with Rauscher murine leukemia virus. For the assessment of the role of peripheral blood mononuclear leukocyte subpopulations in the responsiveness to MuMTV, the cell preparations were fractionated according to their ability to form spontaneous rosettes with sheep red blood cells (E-rosettes). The effectiveness of the separation was ascertained by means of cell surface markers, i.e., presence of surface immunoglobulins or a T-cell marker. Leu-1 antigen, and mitogen-induced blastogenesis. The responsiveness to the MuMTV antigen(s) was associated with the T-cell subset, identified as the E-rosetting. Leu-1-positive, and surface immunoglobulin-negative population. Although some subjects with the normal population gave positive reactions, the results reveal an apparent association between high levels of responsiveness to MuMTV within the T-lymphocyte subset and breast cancer disease.

A multicenter clinical trial of oral ribavirin in HIV-infected people with lymphadenopathy: virologic observations. Ribavirin-LAS Collaborative Group.

A double-blind, randomized, placebo-controlled trial comparing two daily doses of oral ribavirin (600 and 800 mg) and a placebo was performed at four medical centers geographically distributed throughout the USA. One hundred and sixty-four HIV-infected adult men with lymphadenopathy were enrolled over a 2-month period and received active treatment for 24 weeks followed by a 4-week interval during which they did not receive the study drug. A marked interlaboratory variation in HIV isolation from peripheral blood mononuclear cells was observed, underscoring the critical role of quality assurance in similar multicenter trials. Nevertheless, the combined data indicate that ribavirin did not significantly suppress HIV activity (on measurement of reverse transcriptase activity) after week 6 or reduce serum p24 antigenemia.

HIV-1 inhibition by azidothymidine in a concurrently randomized placebo-controlled trail.

Two independent measures of human immunodeficiency virus type 1 (HIV-1) infection, virus isolation, and serum levels of p24 antigen were evaluated in a double-blind randomized clinical trial of the safety and efficacy of a nucleoside analogue, 3'-azido-3'-deoxythymidine (AZT) versus placebo in a single center. Pretreatment studies from 38 AIDS and AIDS-related complex (ARC) patients were comparably positive for virus isolation from their lymphocytes; all patients were qualitatively virus positive. Before AZT treatment, there was significantly decreased virus recovery in patients with higher numbers of CD4-positive lymphocytes. Within 1 month of AZT therapy, the time in culture required to register virus positivity was increased markedly in the AZT-treated group, and over the following several months progressive diminution in virus recovery was noted. Similar changes were not seen in patients concurrently receiving placebo treatment. Before treatment, 16 of 20 and 12 of 16 patients in the AZT and placebo groups, respectively, were p24 antigen positive. Marked reduction in serum p24 levels were noted in 11 of 16 (69%) of the p24 antigen-positive AZT-treated patients compared to 3 of 12 (25%) of the p24 antigen-positive placebo-treated patients (p = 0.02). There was a marked virologic response in 14 of 20 (70%) of the AZT-treated patients compared to 4 of 18 (22%) placebo-treated patients (p = 0.004). A higher frequency of positive clinical and immunological effects also were noted in the AZT-treated patients relative to placebo-treated patients (p = 0.02 and p = 0.06, respectively).

Human T-cell lymphotropic virus infection among blood donors in south Florida. The Transfusion Safety Study Group.

Knowledge of the epidemiologic pattern of human T-lymphotropic virus (HTLV) in the United States is being enlarged by blood donor screening. We tested stored sera from 29,937 donations made in South Florida in 1984-1985. Twenty-three donors were confirmed as seropositive, a prevalence of 0.8 per 1,000 donations. Specificity was supported by serologic retesting and virus culture of 11 donors located for follow-up. Sex- and age-specific prevalences did not differ significantly; blacks, however, accounted for 65% of seropositive donations. Within South Florida, one section of Miami had a prevalence of 4.5 per 1,000 donations, significantly above the 0.1 to 1.1 per 1,000 rates for other parts. An epidemiologic association with known HTLV-I endemic areas could account for most infections; all seven typed isolates were characterized as HTLV-I. Exposures, however, were diverse, sometimes multiple, and had no necessary relationship to personal lifestyle. This finding suggests that sources of infection were varied. Seropositive family members emphasize familial clustering of HTLV-I infection.

Expression and demethylation of germinally-transmitted BALB/c mouse mammary tumor virus DNA in Abelson MuLV B-lymphoid cell lines.

Murine mammary tumor virus (MMTV) RNA expression, DNA organization and DNA demethylation were examined in BALB/c B-lymphoid cell lines produced by transformation with the Abelson murine leukemia virus (AbMuLV). The MMTV DNA sequences in AbMuLV B cell lines, based on restriction mapping with EcoRI, PstI, BglII, BamHI and SacI and molecular hybridization with cloned probes of the MMTV LTR, gag-pol or env gene regions, were identical to the germinally-transmitted MMTV DNA complement of BALB/c mice. Several AbMuLV B cell lines expressed MMTV poly(A+)-RNA at detectable levels. MMTV poly(A+)-RNA for the env gene, 3.8 kb, and the long terminally redundant (LTR) region, 1.7 kb, were detected in some AbMuLV B cell lines. MMTV DNA sequences in the AbMuLV B cell lines were at least partially sensitive to digestion by the methylation-sensitive restriction endonucleases HhaI and HpaII. HhaI-sensitive sites were present in Units I, II and III of the germinally-transmitted MMTV DNA and were localized specifically near the 5' end of the 5' and 3' LTRs of both Units II and III. HpaII-sensitive sites were localized near the 3' end of the 3' LTRs of Units II and III, and at a cellular site 2.1 kbp 5' to the 5' LTR. These observations demonstrate that the germ line MMTV DNA sequences of BALB/c mice are expressed in cells of B lymphocyte origin, and suggest a correlation between MMTV RNA expression and selective demethylation in the LTR regions of germinally-transmitted MMTV DNA sequences.

Statistical methods for the analysis of HIV-1 core polypeptide antigen data in clinical studies.

Levels of HIV-1 core polypeptide were assessed in serum and in lymphocyte cultures obtained from ARC and AIDS patients enrolled in a prospectively randomized, placebo-controlled study of zidovudine (AZT). Because these data have special features uncharacteristic of most laboratory data, a comprehensive account of statistical methods appropriate for their analysis is contained in this paper. Standard methods are described for the analysis of HIV-1 antigen in serum collected repeatedly over time in the same individual. For the analysis of lymphocyte culture data, more sophisticated statistical techniques based on nonparametric survival analysis methods are proposed. Using microcomputer software available upon request and developed to implement this statistical procedure, a significant decline in lymphocyte HIV-1 virus expression was noted between pretreatment and 3 months after the initiation of therapy among AZT-treated patients (p = 0.0017) that was not seen in placebo-treated patients (p = 0.25). Statistically significant between-group differences were also noted in the change from baseline at 3 months in HIV-1 antigen serum data (p = 0.040). We conclude that HIV-1 core polypeptide is an important measure of the antiretroviral activity of AZT and that the demonstrated clinical efficacy of AZT relative to placebo parallels its antiretroviral effect.