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hTid-1, a human DnaJ protein, is a novel cellular target for HTLV-1 Tax. Here, we show that hTid-1 represses NF-kappaB activity induced by Tax as well as other activators such as tumor necrosis factor alpha (TNFalpha) and Bcl10. hTid-1 specifically suppresses serine phosphorylation of IkappaBalpha by activated IkappaB kinase beta (IKKbeta), but the activities of other serine kinases including p38, ERK2, and JNK1 are not affected. The suppressive activity of hTid-1 on IKKbeta requires a functional J domain that mediates association with heat shock proteins and results in prolonging the half-life of the NF-kappaB inhibitors IkappaBalpha and IkappaBbeta. Collectively, our data suggest that hTid-1, in association with heat shock proteins, exerts a negative regulatory effect on the NF-kappaB activity induced by various extracellular and intracellular activators including HTLV-1 Tax.

OBJECTIVES: We measured the pharmacokinetics and safety of zidovudine in pregnant women infected with human immunodeficiency virus and their offspring. STUDY DESIGN: Asymptomatic human immunodeficiency virus-infected women with uncomplicated singleton gestations (28 to 36 weeks) underwent parenteral and oral zidovudine treatment during pregnancy and labor. Maternal and neonatal drug levels were measured at delivery and sequentially for 48 hours. Infants were followed up for 18 months. RESULTS: The total body clearance (26.3 ± 10.1 ml/min/kg), mean terminal elimination phase zidovudine half-life (1.3 ± 0.2 hours), and urinary zidovudine recovery were similar to values in nonpregnant adults. Essentially equivalent zidovudine levels in the mother and neonate at delivery implied little, if any, fetal zidovudine metabolism. The half-life of zidovudine in the neonates was tenfold that of the mother. No significant adverse effects were noted in the infant at birth or on follow-up. CONCLUSIONS: In both mothers and infants the drug appeared safe and well tolerated with no significant hematologic abnormalities.

To examine the sequence diversity of human immunodeficiency virus type 1 (HIV-1) between known transmission sets, sequences from the V3 and V4-V5 region of the envelope gene from four mother-infant pairs were analyzed. The mean interpatient sequence variation between isolates from linked mother-infant pairs was comparable to the sequence diversity found between isolates from other close contacts. The mean intrapatient variation was significantly less in the infants' isolates then the isolates from both their mothers and other characterized intrapatient sequence sets. In addition, a distinct and characteristic difference in the glycosylation pattern preceding the V3 loop was found between each linked transmission pair. These findings indicate that selection of specific genotypic variants, which may play a role in some direct transmission sets, and the duration of infection are important factors in the degree of diversity seen between the sequence sets.

Most infants with pediatric acquired immunodeficiency syndrome and infections with human immunodeficiency virus type 1 (HIV-1) are infected perinatally by their mothers. To determine the proportion of exposed infants who are infected, we conducted a hospital-based prospective study in HIV-1-infected women whose infants were delivered at a single metropolitan hospital in Miami, Fla. A population of uninfected women and their infants was also enrolled and followed longitudinally for 2 years to assess laboratory and clinical measurements. The median follow-up is now 18 months for 82 infants born to HIV-1-infected mothers. The proportion of infected infants in this group is 0.30 (25/82). None of the infants born to 110 HIV-1-seronegative mothers were seropositive. Infected infants were easily distinguished from noninfected infants by virus isolation. No single immunologic or hematologic measure was predictive of infection for all infants at risk for HIV-1 infection who were 6 months of age or younger. As a group, however, infected infants could be distinguished from uninfected index infants by a number of immunologic measures by 6 months of age; the absolute number of CD4 + lymphocytes and the CD4 + /CD8 + lymphocyte ratio were the variables most predictive of infection. As in retrospective studies, clinical disease developed in 80% of infected infants within the first 24 months of life. This study provides documentation of HIV-1 perinatal transmission risk and early correlates of infection in young infants from a single hospital.

Thirty-five children with symptomatic human immunodeficiency virus infection were enrolled in a 12-week, three-center phase I study of intravenous and oral zidovudine therapy. At enrollment the children ranged in age from 5 months to 13 years, with a median age of 3 1/2 years. Twenty-one children (60%) had acquired immunodeficiency syndrome and 14 (40%) had the related complex; 20 children had 9 CD4+ lymphocytes per liter ( 3 ) at entry. Zidovudine was administered in one of three escalating dose regimens. One or two months of intravenous treatment with zidovudine every 6 hours was followed by orally administered drug on the same schedule; zidovudine was infused at 80, 120, or 160 mg/m 2 /dose, and the oral dose was one and one-half times the intravenous dosage. Adverse events were similar to those observed in adults. Neutropenia (absolute neutrophil count 9 /L (750 cells/mm 3 )) occurred in nine patients. The median neutrophil count fell from 2.50 10 9 /L at entry to 1.72 10 9 /L at the end of the study. Anemia requiring transfusion occurred in seven patients; the median hemoglobin level among nontransfused patients decreased from an entry value of 108 to 105 gm/L (10.8 to 10.5 gm/dl). Dosage adjustments were made in 15 patients, in 12 because of anemia or neutropenia.

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.

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.

Members of the f̲-b̲ox p̲rotein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by s̲kp1, c̲ullin, and one of many f̲bps) act as protein-ubiquitin ligases that control the G1/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias β-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25→q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding β-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.

The Nef protein of the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), is a myristylated protein associated with increased viral replication and enhanced pathogenicity. Both the potentiation of T-lymphocyte activation and the enhanced serine-phosphorylation of HIV-1 capsid by Nef correlate with increased viral replication. We report the functional interactions of the Nef proteins with Src kinases. The Nef proteins from HIV-1 and SIV bind to Lck as well as Hck, Lyn, and Fyn. The SH3 and SH2 domains of Lck are sufficient for coprecipitation with non-tyrosine-phosphorylated Nef proteins. The conserved core region of HIV-1 Nef is essential for the interaction with Lck and is also important for enhanced HIV-1 replication in T-lymphocytes. In addition, we show that SIV and HIV-1 Nef proteins are differentially tyrosine-phosphorylated. The kinase-active Lck tyrosine-phosphorylates SIVmac239 Nef but does not phosphorylate HIV-1 Nef. These data suggest that the association of Nef and Lck is central to the enhanced viral replication of HIV-1 and SIV in T-lymphocytes.

For productive replication of human immunodeficiency virus type 1 (HIV-1) in host cells, the viral genome-encoded transactivator Tat and several cellular transcription factors are required for efficient viral gene transcription. However, it remains unclear how the viral genome initiates transcription before Tat is transcribed or when Tat is at suboptimal levels. Here, we utilized the human T-cell leukemia type 1 Tax protein as a molecular tool to investigate the mechanism of viral gene transcription that initiates the early phase of infection of HIV-1. Tax alone does not significantly increase the activity of HIV-1 long terminal repeat (LTR) in T lymphocytes, but it markedly enhanced the replication of an infectious HIV-1 provirus with a truncated nef gene. This enhancement is preferentially mediated by the cooperation of Tax and Tat which is dependent on TAR and duplicated κB cis elements of the HIV-1 LTR as well as the NF-κB activation domain of Tax. Furthermore, phorbol myristate acetate and membrane-targeted HIV-1 Nef also enhanced the LTR activity in the presence of Tat in the TAR- and κB cis element-dependent manner. These data suggest that activated NF-κB can functionally interact with a suboptimal amount of Tat and the HIV-1 LTR for efficient initiation of viral gene transcription.

Human T-cell lymphotrophic virus type 2 (HTLV-2), a common infection of intravenous drug users and subpopulations of Native Americans, is uncommon in the general population. In contrast with the closely related HTLV-1, which is associated with both leukemia and neurologic disorders, HTLV-2 lacks a strong etiologic association with disease. HTLV-2 does shares many properties with HTLV-1, including in vitro lymphocyte transformation capability. To better assess the ability of HTLV-2 to transform lymphocytes, a limiting dilution assay was used to generate clonal, transformed lymphocyte lines. As with HTLV-1, the transformation efficiency of HTLV-2 producer cells was proportionately related to the number of lethally irradiated input cells and was comparable to HTLV-1-mediated transformation efficiency. HTLV-2-infected cells were reproducibly isolated and had markedly increased growth potential compared to uninfected cells; HTLV-2 transformants required the continued presence of exogenous interleukin 2 for growth for several months and were maintained for over 2 years in culture. All HTLV-2-transformed populations were CD2 and/or CD3 positive and B1 negative and were either CD4 + or CD8 + populations or a mixture of CD4 + and CD8 + lymphocytes. Clonality of the HTLV-2 transformants was confirmed by Southern blot analysis of T-cell receptor β chain rearrangement. Southern blot analysis revealed a range of integrated full-length genomes from one to multiple. In situ hybridization analysis of HTLV-2 integration revealed no obvious chromosomal integration pattern.

Human T-cell lymphotropic virus type 1 (HTLV-1) enhances the growth of T lymphocytes, allowing the generation of T-lymphocyte cell lines. This report describes a limiting-dilution assay system which uses low input numbers of HTLV-1-producing cells for generation of T-lymphocyte cultures. The HTLV-1 transformants generated with this assay system produced high levels of HTLV-1 p24 antigen and required exogenous cytokines for maintenance. Clonal populations of CD4- or CD8-positive HTLV-1 transformants were generated with transformation efficiency rates as high as 78%. An exogenous cytokine is necessary for HTLV-1 T-lymphocyte transformation, and cytokine dependence is the most likely outcome of infection and transformation. HTLV-1 T-lymphocyte transformation can occur in the presence of cytokines other than interleukin-2 (IL-2), such as IL-4 or IL-7. IL-4- or IL-7-dependent HTLV-1 transformants underwent T-lymphocyte mitogenesis in response to their homologous cytokines but proliferated best in the presence of IL-2. Since the receptors for IL-2, IL-4, and IL-7 share the IL-2 gamma chain, this component may be the common element in the signaling pathway for HTLV-1-associated transformation.

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.

A quantitative analysis of antibody responses to human immunodeficiency virus type 1 (HIV-1) proteins using Western immunoblots and 125I-labeled protein A is reproducible and can be validated. The antibody levels obtained by Western immunoblots were compared with stoichiometric p24 radioimmunoassay over a wide range of antibody (correlation coefficient, .94; P less than .001). Antibody levels to gp160 and gp120 were validated using purified antigens. Analysis of antibody levels from 31 seropositive individuals revealed a statistically significant correlation between antibody levels to p24 and the other viral proteins except gp120. Anti-gag p24 antibody was strongly correlated with antibodies to other env products, specifically gp41 and gp160. Using the validated assay, HIV-1-infected mothers of infants were found to have highly variable levels of antibody to all viral proteins. Mothers of infected infants did not differ significantly from mothers of uninfected infants in antibody pattern or levels to any viral protein including gp120.

OBJECTIVES: We measured the pharmacokinetics and safety of zidovudine in pregnant women infected with human immunodeficiency virus and their offspring. STUDY DESIGN: Asymptomatic human immunodeficiency virus-infected women with uncomplicated singleton gestations (28 to 36 weeks) underwent parenteral and oral zidovudine treatment during pregnancy and labor. Maternal and neonatal drug levels were measured at delivery and sequentially for 48 hours. Infants were followed up for 18 months. RESULTS: The total body clearance (26.3 ± 10.1 ml/min/kg), mean terminal elimination phase zidovudine half-life (1.3 ± 0.2 hours), and urinary zidovudine recovery were similar to values in nonpregnant adults. Essentially equivalent zidovudine levels in the mother and neonate at delivery implied little, if any, fetal zidovudine metabolism. The half-life of zidovudine in the neonates was tenfold that of the mother. No significant adverse effects were noted in the infant at birth or on follow-up. CONCLUSIONS: In both mothers and infants the drug appeared safe and well tolerated with no significant hematologic abnormalities. (AM J QBSTET GYNECOL 1993;168:1510-6.)

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2,3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2,4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein–protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.

Of 155 children with the acquired immunodeficiency syndrome (AIDS) whom we evaluated during a 6 1/2-year period, 12 were found to have proteinuria. Histologic studies of tissue from these 12 patients revealed a wide spectrum of renal disease: focal glomerulosclerosis in 5, mesangial hyperplasia in 5, segmental necrotizing glomerulonephritis in 1, and minimal change disease in 1. In addition, 6 had tubulointerstitial infiltrates, and 10 had glomerular dense deposits. All 10 renal specimens studied by electron microscopy contained endothelial tubuloreticular inclusions. The mean age (+/- SD) of the five patients with focal glomerulosclerosis when this condition was identified was 27 +/- 19 months. All five had severe renal failure within a year and died of other causes during the following year. The mean age of the five patients with mesangial hyperplasia was 38 +/- 31 months. Although none of them went on to have renal failure, four died within 8 +/- 7 months. Ten of the 12 patients with proteinuria died during the study period. Of the two surviving, one had mesangial hyperplasia and the other had minimal change disease. We conclude that children who acquire human immunodeficiency virus (HIV) infection during the perinatal period may have renal disease, most often focal glomerulosclerosis, as is the case in adults, or mesangial hyperplasia. Although 5 of the 12 children we studied had renal failure during the study period, none died of it. Further studies are needed to determine the correlations between clinical and pathological features and the pathophysiology of AIDS nephropathy in children.

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.

We describe our experience at Jackson Memorial Hospital in Miami, Florida, with 172 children who were given diagnoses of perinatally acquired infection with human immunodeficiency virus type 1 (HIV-1). The 146 mothers of the children acquired HIV-1 through heterosexual contact (69 percent), intravenous drug use (30 percent), or blood transfusion (1 percent). The children presented with symptomatic disease at a median age of eight months; only 21 percent presented after the age of two years. The most common first manifestations of disease were lymphoid interstitial pneumonia (in 17 percent), encephalopathy (in 12 percent), recurrent bacterial infections (in 10 percent), and candida esophagitis (in 8 percent), for which the median survival times from diagnosis were 72, 11, 50, and 12 months, respectively. Nine percent of the children had Pneumocystis carinii pneumonia at a median age of five months and had a median survival of only one month. The median survival for all 172 children was 38 months from the time of diagnosis. Mortality was highest in the first year of life (17 percent), and by proportional-hazard analysis the probability of long-term survival is low. In multivariate analyses, early age at diagnosis and the first identifiable pattern of clinical disease were found to be independently related to survival. We conclude that children with perinatally acquired HIV-1 infection have a very poor prognosis and that most become symptomatic before one year of age. Early diagnosis is important, since there is only a short interval in which to initiate prophylactic or antiviral treatment before progressive disease begins.

Two low-molecular-weight type-specific virion polypeptides from the Kirsten strain of Murine leukemia virus, polypeptides p10 and p12, are immunologically related by radioimmunoassay competition techniques.

A selection system is described for the isolation of somatic cell hybrids which does not require mutagenesis or Budr treatment of either parental cell. Thioguanine-resistant Kirsten sarcoma virus transformed nonproducer rat cells have been fused to contact-inhibited mouse cells and the hybrids selected in soft agar containing hypoxanthine, aminopterin, and thymidine. The somatic cell hybrids obtained by this procedure between mouse and rat cells have been used to study the host range of xenotropic type C viruses. The restriction by murine cells to the growth of certain xenotropic murine type C viruses was found to be dominant in such hybrids.

The viral RNA of the Harvey strain of murine sarcoma virus (Ha-SV), which does not encode for any known viral structural polypeptides, has been translated in a nuclease-digested, cell-free system. The major protein product of the in vitro translation reaction has a molecular weight of 21,000 and is initiated faithfully with [35S]formylmethionine from formyl-[35S]methionyl-tRNAFMET. This polypeptide is clearly distinct from the RNA of the Moloney strain of type C helper virus used to pseudotype the Ha-SV. The intensity of the 21,000-dalton polypeptide on gels correlates well to the concentration of Ha-SV RNA in different viral RNA preparations. These experiments indicate that a polypeptide marker for Ha-SV is now available for the first time. The possibility that this protein is the product of the rat portion of the Ha-SV genome is discussed.

Template active chromatin and template inactive chromatin have been fractionated from mouse cells infected with the Moloney strain of murine leukemia virus. In vivo the cells produce abundant RNA homologous to Moloney leukemia virus, but do not produce either globin mRNA or RNA homologous to type B mouse mammary tumor virus. The DNA extracted from the template active chromatin or template inactive chromatin contained equal amounts of sequences homologous to Moloney type C virus, to type B virus, or to globin mRNA. The results are discussed with regard to the in vivo structure of chromatin and the difficulties in fractionating chromatin in vitro.

The specificity and quantitation of the rescue of RNA sequences by mammalian type C viruses has been investigated. Type C viruses can package with specificity only type C viral RNA. Type C viruses do not encapsidate with comparable efficiency either type B viral or cellular globin mRNA. Conversely, a non-type C mammalian retravirus, MP-MV, cannot encapsidate type C RNA. A revertant of Kirsten sarcoma virus (Ki-SV)-transformed nonproducer cells which fails to rescue biologically active Ki-SV after superinfection with helper virus had no detectable intracellular Ki-SV -specific RNA. The results suggest specific mechanisms by which type C viral proteins can package type C viral RNA and provide an approach to classifying RNA of potentially defective endogenous retraviruses as type C in origin.

A review of the genetics of mammary tumor virus is presented in which the discoveries and hypotheses are given as they have historically developed.

Major virion low-molecular-weight polypeptides were isolated from the Moloney strain of murine leukemia virus (type C) by agarose chromatography in 6M guanidine hydrochloride and were shown to have molecular weights of 15,000 (p15), 12,000 (p12), and 10,000 (p10) by their elution volumes and by their relative mobilities in sodium dodecyl sulfate-polyacrylamide gels. Each polypeptide could be iodinated and employed in double antibody radioimmunoassay procedures. All three polypeptides demonstrated a high degree of type-specificity in serologic immunoprecipitation analysis and in corresponding competition immunoassays. The p15 was immunologically distinct from other viron polypeptides including p12 and p10; the p12 and p10 were highly related to each other but not to other virion polypeptides and were even more type-specific than the p15 in serologic tests. Competition immunoassays with p15 and p10 indicate that the Moloney strain of MuLV is only a distant relative of the Friend-Rauscher group. The combined use of the Kirsten and Moloney low-molecular-weight polypeptide immunoassays suggest that xenotropic viruses constitute yet another group(s) of murine leukemia virus with distinct type-specific antigens, further expanding an already heterogeneous group of mouse type C viruses.

Four adults form four separate households were found to have simultaneous retroviral infections with human T cell leukemia virus type I (HTLV-I) and human immunodeficiency virus (HIV). These individuals were seropositive for the HTLV-I env transmembrane protein p21E, and all had antibodies to the HTLV-I core polypeptide p24. All four patients also had antibodies to the HIV env transmembrane polypeptide p41E and to the HIV core polypeptide p24. HTLV-I was isolated from peripheral blood lymphocytes of all four individuals, and both viruses were isolated from two of them. Evidence of HIV transmission was noted in the family contacts. Eight of 10 children of these four adults were seropositive for HIV, presumably because of perinatal transmission from infected mothers. Two of five spouses of these adults were examined; these spouses had antibodies to HIV and were positive for virus. No evidence of HTLV-I transmission was noted in these families.

The nucleic acid sequences found in the Harvey strain of murine sarcoma virus have been analyzed by RNA·[ 3 H]DNA and [ 3 H]RNA·DNA hybridization techniques. The Harvey strain of murine sarcoma virus has been found to possess at least two sets of nucleic acid sequences. One set of sequences is contained in the Moloney strain of mouse type-C virus, and the other set is contained in DNA transcripts synthesized in endogenous reactions containing rat type-C virus(es). The nucleic acid sequences that are detected in the Harvey sarcoma virus with the DNA probes synthesized from the rat type-C virus(es) are related to the rat sequences detected in the Kirsten strain of murine sarcoma virus. The results support the model that both Kirsten and Harvey sarcoma viruses arose through a process of recombination or reassortment between mouse type-C viruses and sequences in rat cells and suggest that the information for transformation of fibroblasts may be contained in the rat type-C or cellular genome.

The high prevalence of human immunodeficiency virus (HIV-1) infection in populations at risk in Miami prompted a seroepidemiologic study of both HIV-1 and the human T -cell leukemia virus type I (HTLV-I), a closely related virus, in our patients receiving chronic hemodialysis. One hundred twenty-nine patients undergoing hemodialysis in 1986 and 1987 were tested for antibody against both viral antigens by EIA (Abbott Laboratories, Abbott Park, IL). Seroreactive samples for HIV-1 and/or HTLV-1 were confirmed by Western blot and, for HTLV-I, by viral cultures. Thirty patients (23.2%) were positive for retroviral infection (22 for HIV-1 alone, four for HTLV-1 alone, and four for both HIV-1 and HTLV-I). The most important risk factor was intravenous drug use, followed by blood transfusion. Patients with HIV-1 had lower T 4 -T 8 ratios and higher mortality than those with HTLV-1 infection alone. It was concluded that HTLV-I, as well as HIV-1, infection is endemic in chronic dialysis centers in Miami. The clinical consequences of HTLV I infection in relatively immunocompromised patients with chronic uremia who are undergoing chronic hemodialysis remains to be established.

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.

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.

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.

Genotypic variation among independent isolates of human immunodeficiency virus type-1 (HIV-1) is well known, but its molecular basis and biological consequences are poorly understood. We examined the genesis of molecular variation in HIV-1 by sequential virus isolations from two chronically infected individuals and analysis of recombinant HIV-1 genomic clones. In three different virus isolates full-length HIV-1 clones were identified and found to consist, respectively, of 17, 9 and 13 distinguishable, but highly-related, viral genotypes. Thirty-five viral clones derived from two HIV-1 isolates obtained from the same individual but 16 months apart showed progressive change, yet were clearly related. Similar changes in the HIV-1 genome did not occur in vitro during virus isolation and amplification. The results indicate that HIV-1 variation in vivo is rapid, that a remarkably large number of related but distinguishable genotypic variants evolve in parallel and coexist during chronic infection, and that 'isolates' of HIV-1, unless molecularly or biologically cloned, generally consist of complex mixtures of genotypically distinguishable viruses.

An isotopic antiglobulin assay was developed to measure antibodies in “normal” cats to FOCMA, a cell surface antigen associated with immunity to FeLV-induced tumors. The assay was shown to be specific for detecting FOCMA antibody by several criteria including a correlation with cell membrane immunofluorescence and absorption tests. Antibody to FOCMA developed following FeLV infection and was expressed in heterologous species. In combination with an assay for antibody to FeLV p30, the major virion structural protein, 200 cat sera from three different geographic areas were examined for evidence of FeLV type-C virus infection. Approximately 17% of all sera examined contained antibody to FOCMA. Antibodies directed to the FeLV p30 were detected in 8 of the sera and only in cats with FOCMA antibody. The correlation in cat sera between antibody to FOCMA and FeLV p30 antigen suggests that both are associated with the same process, infection with FeLV type-C virus. The relationship of the observed pattern of antibody response is discussed in relation to other horizontally transmitted RNA-containing viruses.

SummaryPrevious studies demonstrated that despite freedom from MTV and mammary tumors early in life Balb/cCrgl mice possess lymphocytes sensitive to mouse mammary tumor antigen(s) within 2-3 weeks of life. This sensitivity was manifested in blastogenic transformation and migration inhibition reactions using RIII milk and purified MTV from RIII milk. These reactions were not elicited with the MTV-negative milk from NIH Swiss mice. Preincubation of MTV with anti-MTV antibody inhibited the blastogenic reaction. In contrast, antibody raised against a type C murine oncornavirus had no inhibitory effect and did not by itself induce blastogenic transformation. The nature of the virus-related antigen responsible for the lymphocyte sensitivity has not been ascertained, nor has its location in the animals' tissue been determined. However, radioimmune assays for p14 antigen in multiple tissues of Balb/c mice have yielded insignificant values suggesting either incomplete expression of the genome and/or different leve...

Cats which have been infected with FeLV and which have recovered without disease or persistent viremia are at a low to negligible risk for subsequent development of FeLV-associated neoplasia. To evaluate the immunologic correlates of this protection, cat sera from various free-living populations were shown by immunoprecipitation of FL74 cell surface proteins to react with an 85,000 dalton cell surface protein, p85. All sera from non-viremic cats with FL74 cell membrane immunofluorescence reactivity greater than 1:32 precipitated p85. The reactivity of p85 and concomitant cell membrane reactivity could be completely removed from serum with freshly purified, undisrupted FeLV and could be blocked with specific antisera to known virion envelope glycoproteins, demonstrating that p85 contains determinants of the FeLV envelope glycoprotein. The reactivity to p85 could also be removed with FeLV-infected feline and nonfeline cells but not with cells nonproductively transformed by the feline sarcoma virus. These data strongly suggest that a major cell membrane antibody in feline sera is directed to the major FeLV envelope glycoprotein expressed on the surface of cells infected with FeLV but not FeSV. The relationship of this reactivity to the previously and presently defined anti-“FOCMA” reactivity is discussed.

A major murine mammary tumor viral (MMTV) antigen, sl, originally described by Nowinski et al. (1967, 1968, 1971), has been purified from RIII mouse milk MMTV by sequential ion-exchange and gel chromatography. The purified protein with sl antigenic reactivity contains carbohydrate, and has an apparent minimal molecular weight of 52,000. It can be designated as gp52 (sl). Another major MMTV viral protein with a molecular weight of 27,000 has also been isolated, and antisera have been prepared against it. Both MMTV gp52 (sl) and p27 viral polypeptides have been iodinated with 125 I and used in immunoprecipitation and competition assays. The two MMTV proteins differ absolutely from each other and from major mouse type C viral polypeptides in molecular weight, immunological reactivity, and amino acid composition. Purified gp52 (sl) in radioimmunoprecipitation inhibition assays reacted in two distinct patterns. One pattern showed partial displacement of antibody which could be converted to the second, a complete displacement, by heating the antigen, presumably by exposing additional reactive determinants. Biologically, the patterns of major MMTV polypeptide expression in milk correlated with spontaneous mammary tumor incidence in different strains of mice, indicating that the sl antigen is group specific for MMTV or that several mouse strains contain the same virus type.

A 14,000-dalton polypeptide (p14) from RIII murine mammary tumor virus (MMTV) has been isolated by column chromatography in 6 M GuHCl. Antiserum prepared in rabbits specifically precipitated 125I-labeled p14; in double antibody competition, radioimmunoassays performed with limiting amounts of antibody, both purified p14 and disrupted MMTV, competed specifically with labeled antigen. The expression of this MMTV type B virus antigen could be measured by competition radioimmunoassays in milks, mammary glands, tumors, and tissue culture cells. MMTV expression measured by p14 immunoassay correlated well with the spontaneous incidence of mammary adenocarcinomas in different murine strains but not with type C MuLV p30 antigen expression. Levels of MMTV gp52, the major type-B viral glycoprotein, corresponded to p14 levels, suggesting that their control is comparably regulated. Evidence that this low m.w. polypeptide is present in feral and inbred strains of widely differing geographic origin and in MMTV with apparently different biologic properties suggests surprising conservation of MMTV protein homology.

In a study being reported concurrently, we confirmed the observation of Deinhardt et al. [1] that a pool of fifth passage sera from marmosets originally inoculated with human hepatitis material was transmissible in marmosets and produced a hepatitislike picture [2]. That study characterized the infection in marmosets following inoculation with the Barker strain of marmoset hepatitis virus and reported some physical characteristics of the agent. Histological studies demonstrated in the livers of infected animals a characteristic inflam

The study of human viral hepatitis has been hampered by lack of susceptible tissue cultures or animals [1, 2]. An encouraging lead was recently obtained by Deinhardt et al. [3] who inoculated human sera into marmosets and obtained from the serum of certain inoculated animals a transmissible agent that produced histologic hepatitis associated with a rise in serum transaminase. Their initial report dealt with the isolation of the virus from human materials and with the clinical and histological features of the infection in marmosets. Hepatitis was not found in control animals in their study. A brief report [4] noted that commercial pooled human IgG strikingly reduced the infectivity of infectious marmoset sera, but this could not be confirmed in later experiments [5]. Using infectious marmoset sera kindly supplied by Deinhardt and Holmes, we have confirmed the existence of a transmissible hepatitis in marmosets and have investigated the physical properties of the virus and the characteristic features of the infection.

A clonal isolate of an RNA-containing sarcoma virus derived from a spontaneous woolly monkey fibrosarcoma has been obtained. The woolly sarcoma virus transformed cells do not spontaneously yield infectious virus but do contain the woolly gs antigen. The primate sarcoma virus can be rescued with comparable efficiency by superinfection with either murine or primate helper type-C viruses. During rescue with murine helper type-C virus, the intracellular level of woolly monkey gs antigen does not rise in proportion to the production of helper gs antigen, or in proportion to the production of transmissible focus-forming virus. The results suggest that the mechanism of rescue may simply involve an assembly of sarcoma components into an infectious particle during the production of the helper type-C virus.

A radioimmune precipitation assay (RIPA) for the major internal polypeptide of mammalian type C viruses is described. Gel chromatography and isoelectric focusing resulted in a polypeptide of approximately 30,000 daltons which migrated as a single band in three different dissociating systems as analyzed by polyacrylamide gel electrophoresis. This VP3(gs) protein from murine and feline type C viruses has species specific antigenic reactivities which were not altered by chemical iodination. 125I-labeled VP3(gs) was a sensitive probe for antibodies. By employing limiting concentrations of antibody in RIPA, unlabeled murine or feline VP3(gs) antigen could be quantitated by competition with labeled polypeptide. This double antibody VP3(gs) antigen assay was at least 500 times more sensitive than complement-fixation or immunodiffusion and was specific for type C viruses of the homologous species.

Based on protein, deoxyribonucleic acid (DNA), and phosphorus analyses, adeno-associated satellite virus type 4 has a DNA content of 26.5%, significantly larger than type 1 which contains 18.9% DNA. Type 4 DNA contains 58 to 62% guanine + cytosine, as calculated from thermal denaturation temperature and buoyant density measurements. Simian adenovirus SV15, which serves as a helper virus for type 4 satellite, contains 13.7% DNA with a guanine + cytosine content of 55 to 57%. The DNA extracted from type 4 satellite virus is a linear, double-stranded molecule, as shown by thermal denaturation temperature profile measurements and by electron microscopy. The contour length and sedimentation coefficient of type 4 DNA indicate a molecular weight of 3.0 × 10 6 daltons.