Purification and characterization of a factor which inhibits retrovirus replication.

Chick embryo cells (CEC) secrete a factor which inhibits HIV1 replication. Non-productive infection of CEC with vesicular stomatitis virus increases 5-10-fold the synthesis of this factor. After purification by FPLC the biological activity is associated with a heat-resistant protein of approximately 150 kDa, composed of two subunits of 70 and 58 kDa and charged negatively. Monoclonal antibodies raised against this protein block the inhibitory activity of the purified protein and react with the 150-kDa and 58-kDa proteins in Western blots.

Effect of retinoic acid on HL-60 cells infected with human immunodeficiency virus type 1.

HL-60 cells infected with human immunodeficiency virus type 1 (HIV 1) can be induced to differentiate along the granulocyte pathway by retinoic acid. In these cells, HIV mRNA synthesis is stimulated, but synthesis of viral proteins and virus replication are blocked and HIV-infected cells die after becoming apoptotic and/or vacuolized.

Virucidal effects of glucose oxidase and peroxidase or their protein conjugates on human immunodeficiency virus type 1.

Glucose oxidase and peroxidase (lactoperoxidase or myeloperoxidase) are virucidal to human immunodeficiency virus type 1 (HIV-1) in the presence of sodium iodide, as assessed by the loss of viral replication in a syncytium-forming assay or by the inhibition of cytopathic effects on infected cells. In the presence of low concentrations of sodium iodide, five HIV-1 isolates were equally susceptible to this virucidal system at enzyme concentrations of a few milliunits. The loss of viral replication was linearly related to the time of incubation in the enzyme solutions, with an inactivation rate of 1 log unit every 30 min. These enzymes and this halide were also cytotoxic to chronically infected, but not to uninfected, cultured CEM cells. Protein conjugates were prepared by using the enzymes and murine antibody 105.34, which recognized the V3 loop of HIV-1 LAI isolate surface glycoprotein, or recombinant human CD4. The protein conjugates inactivated free virus at rates similar to those of the free enzymes and were more effective than antibody or recombinant CD4 alone. These in vitro findings demonstrate that the peroxidase-H2O2-halide system provides potent virucidal activity against HIV-1.

Replication of human immunodeficiency virus in HL-60 cells.

HL-60 cells carrying the CD4 marker could be productively infected with human immunodeficiency virus (HIV) but did not form syncytia, though 11-14 days p.i., there was a transient decrease in cell multiplication and viability. After prolonged passage, a subpopulation of HL-60 cells was selected. The virus produced differed from the initial input virus grown on CEM cells: the virions lacked knobs, were either empty or had abnormal cores, had a higher ratio p24/gp41,gp110 and were less infectious. After prolonged passage, virus was produced which was fully infectious for CEM but not for fresh HL-60 cells, and which ressembled the input virus with respect to morphology and p24/gp41,gp110 ratio.

Inhibition of human immunodeficiency virus (HIV) type 1 multiplication by an avian cellular factor.

A factor secreted from avian cells infected non productively with a non cytopathogenic mutant of vesicular stomatitis virus (VSV ts 1026) interferes with HIV replication in CEM cells and peripheral blood monocytes (PBL). Production of infectious particles is decreased and many virions lack cores and/or spikes. In CEM cells the prmRNA is spliced into 7.5, 4, and 2 kb mRNA. Residual virus contains less env encoded proteins and p 18; p 25 appears as several bands. The processing of tat, rev. and nef proteins differs in treated cells and in controls.

Teacher perceptions of the regular education initiative.

A survey of 381 special and regular educators assessed perceptions and opinions surrounding the regular education initiative. Confirmatory factor analysis supported an a priori hypothesized structure of teachers' responses. Items factored into 14 categories. These factors describe issues related to preferred placement of students with mild disabilities, teachers' responsibility and ownership, teacher preparedness for meeting the needs of these students, achievement outcomes for all children, and the changes that would result from adopting the proposed consultant model rather than a pullout program. Results favored current special education practices (pullout programs) in elementary schools.

Quantification of alphafetoprotein (AFP) endocytosis by PHA-activated peripheral blood mononuclear cells (PBMC) from HIV-infected individuals. A useful test of predictive value for the progression of AIDS.

Serum alphafetoprotein (AFP) is actively taken up, through receptor-mediated endocytosis, by many embryo-fetal cells during ontogenic development but also by neoplastic cells, as well as by normal peripheral T lymphocytes after mitogenic activation. We have previously shown that the ability to internalize AFP is impaired in mitogen-activated T cells from several groups of (HIV+)-seropositive individuals and that this expression roughly correlates with the progression of the disease. It is not clear whether this impaired AFP-endocytosis results from an HIV-mediated inhibition of AFP-receptor expression or if it is the consequence of a target signal transduction impairment due to HIV-infection. In the present work we have explored both possibilities by studying AFP-endocytosis in peripheral blood mononuclear cells (PBMC) from seropositive (HIV+)-asymptomatic heterosexual individuals and in in vitro HIV-infected PBMC from healthy donors, either quiescent or stimulated with phytohemagglutin (PHA). Our results suggest that this novel abnormality of T-cells associated with HIV-infection reflects an unusual proliferative response of PBMC to mitogenic stimuli.

Changes in lipid composition of human peripheral blood lymphocytes infected by HIV.

The possible differences in lipid composition between human immunodeficiency virus- (HIV) infected and uninfected PHA-activated human peripheral blood mononuclear cells (PBMC) have been studied. The total fatty acid composition was similar, except for the proportion of arachidonic acid, that was slightly higher in infected than in noninfected cells. No significant differences were obtained in the incorporation of radiolabeled stearic or oleic acids in the different lipid classes. The staining of cells with Nile Red showed similar amounts of intracytoplasmic lipid droplets. On the contrary, the CH/PL ratio, the major factor in determining cell membrane fluidity, was clearly higher in infected than in uninfected cells (0.60 and 0.36, respectively). This fact is discussed in relation with the known high CH/PL ratio (0.95) of the lipid envelope of HIV.

Purification and characterization of proteins with associated tyrosine protein kinase activity from human B lymphocytes.

Burkitt lymphoma cells and their counterpart of normal origin contain proteins with associated tyrosine protein, kinase activity. These proteins were isolated by affinity chromatography and Fast Pressure Liquid Chromatography. Proteins with enzyme activity had an app. M. W. of 47 KDa. This protein in extracts of Burkitt lymphoma cells differed by overall charge and phosphorylation from the 47 KDa protein isolated from B lymphocytes of normal origin. Before and after purification the 47 KDa protein of Burkitt lymphoma cells reacted with an antibody directed against the dodecapeptide Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg (conserved region of pp60src), the 47 KDa protein from B cells of normal origin did not; the same protein from both cell lines reacted with anti-pp60src antibody. These results suggest that a tyrosine protein kinase, related to the products of the src family of oncogenes, is modified in Burkitt lymphoma cells.

Viral products in cells infected with vesicular stomatitis virus and superinfected with Rous sarcoma virus.

In cells infected with Vesicular Stomatitis virus (VSV) ts 1026 and superinfected with Rous Sarcoma virus (RSV) synthesis of vsrc mRNA and RSV env mRNA decreases. In these cells post-translational processing of RSV precursor proteins is impaired and small amounts of VSV antigens are detected.

Differentiation of Burkitt lymphoma cells by hexamethylenbisacetamide.

Hexamethylenbisacetamide (HMBA) can induce the Burkitt lymphoma Raji cells to enter the differentiation process as evidenced by the decrease of HLA-DR antigens. This event is preceded by a decrease of c-myc expression and of the phosphorylation of cellular proteins, due to either a decrease of tyrosine protein kinase activity or an increase of tyrosine phosphatase activity. These three events form a sequence and are part of the genetic program for differentiation and growth though they may not be causally related.

Integration and expression of provirus in human cells transformed by avian sarcoma virus.

Previously, human diploid fibroblasts from some donors infected in vitro by avian sarcoma virus (ASV) were transformed and found, by electron microscopy, to produce small numbers of virus particles that were infectious by bioassay; also, a line of human osteosarcoma cells infected with ASV developed additional characteristics of transformation and released a small number of infectious virus particles. In this study the complete proviral sequence was shown to be integrated in the genome of these cells. The env-related proteins gp85 and gp37 and the gag-related proteins pr76, pr60, and p19 can be detected in cytoplasmic extracts of ASV-infected human cells. Comparable amounts of pp60v-src were found in human and avian cells infected with ASV. The associated kinase activity in infected human cells was dramatically increased as compared to that of uninfected controls; the enzyme had the same cation and substrate requirements as those from ASV-transformed avian cells. Replicating particles from infected human cells were purified and were significantly modified compared to those from avian hosts as shown by a) higher specific gravity, b) the presence of RSV gag-related but not env-related antigens, and c) the fact that the virus-associated reverse transcriptase preferred the divalent cations Mn2+ and Fe2+ over Mg2+.

Binding of proteins, including pp60src, to activated CH-sepharose 4B.

Activated CH-Sepharose 4B and protein A Sepharose CL-4B can bind, selectively and non-specifically, polypeptides from chick embryo cells. The major polypeptides bound have apparent molecular masses of 57-60 kDa and 47-49 kDa and cannot be eluted by extensive washing with buffers containing detergents. One of the 57-60 kDa polypeptides was identified by immunoblotting as the transforming protein of Rous Sarcoma Virus (RSV), pp60src. This polypeptide could be removed from the solid phase immunoabsorbent with 60% dimethylsulfoxide, but not with 2% SDS, 5% beta-mercaptoethanol, 1 M NaCl or 0.1% Tween 20.

Isolation of proteins with kinase activity and related to pp60 src from human cells.

A protein kinase activity (PK) was associated with immunoprecipitates between polypeptides of human lymphoblastoid cells of malignant origin (Raji cell line) or of their normal counterparts ( Priess cell line) and antibodies directed against avian pp60 src or against the carboxyterminal hexapeptide of pp60 src. Therefore, these human cells and Rous Sarcoma Virus (RSV) transformed avian cells share antigenic determinants of pp60 src and, in particular, its carboxyterminal sequence, as well as one of its functions, a protein kinase activity. The protein kinase from Raji cells phosphorylated predominantly tyrosine residues, that from Priess cells threonine residues.

Inhibition of Rous sarcoma virus-induced transformation by preinfection with rhabdoviruses.

In vivo preinfection of chicks with rabies virus (RV) or vesicular stomatitis virus (VSV) ts 1026 inhibits tumour formation after superinfection with Rous sarcoma virus (RSV). The degree of inhibition depends on the titre of the infecting viruses and the interval between rhabdovirus and RSV infection. In vitro, cells preinfected with VSV ts 1026 under non-permissive conditions and superinfected with RSV, are not transformed as judged by cell morphology, serum requirement for growth or the capacity to form colonies in soft agar, all these being the same as in uninfected cells. Doubly infected cells take up less deoxyglucose than cells infected with RSV only and more than cells infected with VSV only. RSV multiplication in inhibited in doubly infected cells: the supernatant fluid of these cells contains fewer focus-forming units and less reverse transcriptase activity than that of cells infected with RSV only. Doubly infected cells contain both VSV and RSV internal antigens 15 days after infection. The supernatant fluid of cells infected with VSV and maintained under non-permissive conditions inhibits transformation by RSV and multiplication of RSV, but not of VSV. Under non-permissive conditions, the rhabdoviruses undergo at least part of the infectious cycle, but no infectious virus is produced. RV antigen can be detected in the brain of parenterally infected chicks and VSV antigen in cells infected 15 days previously. We conclude that the inhibition of RSV multiplication and expression is probably due to one or more processes linked to the persistence of rhabdovirus components and that it cannot be attributed exclusively to interferon.

Modification of lipophilic proteins in Friend erythroleukemia cells during their differentiation.

In mouse erythroleukemia cells (MELC) a lipophilic protein of apparent M.W. 9.5 kdaltons increases during differentiation. This increase is due either to an increase of biosynthesis or to a structural alteration impairing the capacity of the protein to form polymers of apparent high M.W. or favoring its extractability. The increases is related to differentiation and precedes hemoglobin synthesis by at least 1 day. It is not related to virus production because it occurs in cells (line F 4-1) which do not produce virus, but it does not occur in cells (TFA-II) in which dimethylsulfoxide (DMSO) causes an increase in virus production. As it occurs in cells treated with 4 different inducers, and as the increase is less marked when antagonists of the inducers are also present, it is unlikely that the increase of the 9.5 Kdalton protein is due to an effect of the inducers unrelated to differentiation.