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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An amphotropic retrovirus packaging cell line was constructed in which the gag, pol, and env genes of the helper virus are separated on two different plasmids and in which the packaging signals and 3' long terminal repeats are removed. To do this, a plasmid containing the Moloney murine leukemia virus gag and pol gene was transfected into NIH 3T3 cells, and a plasmid containing the 4070A amphotropic env gene was transfected into one of the resulting clones which produced a high level of reverse transcriptase. A clone producing a high level of amphotropic env protein (GP + envAm12) was then isolated. When transfected into GP + envAm12 cells, titers of the retroviral vector N2, containing a neomycin resistance gene, ranged from 10(2) to greater than 10(6) CFU/ml on 3T3 cells, from 1.3 x 10(4) to 2.7 x 10(5) CFU/ml on HeLa cells, and from 1.0 x 10(2) to 6.0 x 10(3) CFU/ml on K562 cells when assayed by G418 resistance. These titers were comparable to titers obtained using the PA317 cell line. Tests for the safety of the GP + envAm12 packaging line showed no evidence for the generation of wild-type virus. Thus, the efficiency and safety of the GP + envAm12 cell line in gene transfer into human cells may provide an optimal system for experiments whose goal is human gene therapy.
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PMID:Construction and use of a safe and efficient amphotropic packaging cell line. 246 7

The mature product of the env gene of Friend spleen focus-forming viruses (F-SFFV) is efficiently released from both leukemia cells and infected fibroblasts. Analyses of the kinetics of env protein synthesis and secretion in NRK cells infected with the Lilly-Steeves strain of SFFVp indicated that this product, gp65, was formed rapidly and remained stably associated with cells for up to 4 hr, at which point it was first detected in supernatant medium. By 12 hr after synthesis, greater than 95% of gp65 was found extracellularly. The release of this component was effectively blocked by 10 mM 1-deoxynojirimycin, an inhibitor of oligosaccharide processing, demonstrating a requirement for processing of high mannose precursor oligosaccharides in the secretion of gp65. Similar oligosaccharide substituents were found on cell-associated and extracellular forms of gp65. Enzymatic deglycosylation experiments demonstrated that in addition to the predicted four N-linked oligosaccharides, gp65 contains O-linked carbohydrates which are resistant to the action of peptide N-Glycanase F, but sensitive to neuraminidase and O-Glycanase. These structures may be related to O-linked oligosaccharides previously found on the env gene products of murine leukemia viruses. Comparison of the sizes of the deglycosylated forms of cell-associated and supernatant gp65 demonstrated that the extracellular molecules are approximately 3 kDa smaller than the cell-associated components. These data suggest the involvement of proteolysis at a C-terminal site in the release of gp65 from the plasma membrane.
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PMID:Biochemical characterization of cell-associated and extracellular products of the Friend spleen focus-forming virus env gene. 255 67

A synthetic gene for a 88 amino acid long env protein fragment of the human T-cell leukemia virus type 1 (HTLV1) has been assembled by ligation of 35 oligodesoxyribonucleotides, which were chemically synthesized by the phosphotriester segmental support method. After cloning into the pEX vector this HTLV1 env-protein fragment was expressed in E. coli.
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PMID:Chemical-enzymatic synthesis, cloning and expression of a synthetic gene coding for an env protein fragment of the human T-cell leukemia virus type 1. 282 30

A retrovirus packaging cell line was constructed by using portions of the Moloney murine leukemia virus in which the gag, pol, and env genes of the helper virus were separated onto two different plasmids and in which the psi packaging signal and 3' long terminal repeat were removed. The plasmid containing the gag and pol genes and the plasmid containing the env gene were cotransfected into NIH 3T3 cells. Clones that produced high levels of reverse transcriptase and env protein were tested for their ability to package the replication-defective retrovirus vectors delta neo and N2. One of the gag-pol and env clones (GP+E-86) was able to transfer G418 resistance to recipient cells at a titer of as high as 1.7 X 10(5) when it was used to package delta neo and as high as 4 X 10(6) when it was used to package N2. Supernatants of clones transfected with the intact parent gag-pol-env plasmid 3P0 had comparable titers (as high as 6.5 X 10(4) with delta neo; as high as 1.7 X 10(5) with N2). Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus. These results suggest that gag, pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer.
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PMID:A safe packaging line for gene transfer: separating viral genes on two different plasmids. 283 75

Gene expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by two trans-acting factors encoded by the pX region, p40tax and p27tax.p40tax is a transcriptional activator and p27rex is a post-transcriptional regulator. Using full-length viral DNA, we studied the regulatory effects of rex on HTLV-1 gene expression. p27rex is required for expression of both gag and env proteins, increasing the level of their mRNAs. The effect was dependent on the dose of p27rex expression plasmid. In parallel, increased doses of p27rex suppressed the expression of fully spliced pX mRNA, which encodes the regulatory proteins. These two effects of p27rex operated at the post-transcriptional level and were independent of transcriptional regulation. Lowering the level of pX mRNA down-regulates transcription of the proviral genome. These observations demonstrate that rex is a positive post-transcriptional regulator for gag, pol and env protein expression, and acts at the same time as an indirect negative regulator of viral transcription.
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PMID:Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins. 283 30

Friend spleen focus-forming virus (F-SFFV) encodes a glycoprotein designated gp52, which is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum. Only 3-5% of the mature form of gp52 eventually reaches the cell surface. Compared to transport-competent murine leukemia virus (MuLV) glycoproteins, the gp52 molecule exhibits several structural differences which may have resulted in the possible loss of signals required for transport to the cell surface. To determine the effect of these alterations on the specific sites of surface expression of the molecule, the SFFV env gene was expressed from a vaccinia virus recombinant in a polarized epithelial cell line in which retrovirus glycoproteins are expressed exclusively on basolateral surfaces. We also determined the site of expression of a chimeric env protein which contains the external domain of SFFV gp52 the transmembrane, and the cytoplasmic tail residues of Friend MuLV. The wild-type and chimeric env gene products were defective in transport, and remained primarily in an unprocessed form in MDCK cells or CV-1 cells. However, both glycoproteins were detected at low levels on the basolateral surfaces of MDCK cells, a line of polarized epithelial cells. These results indicate that the presence or absence of a cytoplasmic tail as well as a 585-base deletion in the external domain has no affect on the site of polarized expression of a murine retrovirus glycoprotein.
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PMID:Expression of the spleen focus-forming virus envelope gene in a polarized epithelial cell line. 283 65

There is increasing evidence for the link between members of the human T-lymphotropic virus family and clinically important disease. We used indirect membrane immunofluorescence (IMI) to screen patient and control sera for antibodies to human T-cell leukemia virus (HTLV) specific cell membrane antigens (HTLV-MA) of HTLV-I and HTLV-III. Representative sera were screened for antibodies to specific HTLV-encoded proteins using radioimmunoprecipitation (RIP) with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Essentially all Japanese patients with adult T-cell leukemia/lymphoma (ATLL) from Miyazaki, Japan had detectable antibodies to HTLV-I-MA, further supporting the evidence for the probable etiologic relationship of HTLV-I and ATLL. While 16% of the healthy adults from this endemic region had antibodies to HTLV-I-MA, such antibodies were also found in 42% of the adults hospitalized in Miyazaki with severe infections diseases. Other studies have demonstrated HTLV-I antibodies in 12% of asymptomatic hemophiliacs examined from various U.S. cities. We have previously shown that HTLV-I status positive antibody in hemophiliacs is accompanied by a decrease in the number of T helper cells. Patients seropositive for antibodies to HTLV-I-MA regularly demonstrated antibodies to the env gene encoded gp61 proteins, while lower but significant proportions had antibodies to the gag and lor gene proteins. These and other observations suggest that infection with at least some strains of HTLV-I may be associated with mild or transient immunosuppression, in the absence of leukemia. Analysis by RIP indicated that gp61 and gp45, both encoded by the env gene of HTLV-I, are the most immunogenic proteins of the virus. The gp61 HTLV-I is highly crossreactive with gp67, the major env protein of HTLV-II. Patients with acquired immune deficiency syndrome (AIDS) were also examined for antibodies to HTLV-I-MA and antibodies to the gag, env, and lor gene proteins by RIP. Antibodies were detected in 38-75% of the patients, the higher percentage reflecting the presence of at least one positive sample in those individuals where more than three serial serum samples were tested. Numerous control groups were essentially seronegative for antibodies to HTLV-I proteins. When AIDS patient sera were examined for antibodies to HTLV-III, 95-100% were seropositive. Such antibodies were also found in the majority of asymptomatic Boston-areas hemophiliacs.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:HTLV and immunosuppression. 610 Jun 49

A group of temperature sensitive mutants of Moloney murine leukemia virus (MoMuLV) designated as ts1, ts7, and ts11 rapidly and invariably induce hind-limb paralysis ranging from 28 to 52 days postinjection of neonatal CFW/D mice. These temperature-sensitive mutants are defective in the processing of the precursor of the env protein, gPr80env in infected cells, resulting in the accumulation of gPr80env in the infected cell and production of virions with reduced amounts of gp70, p15E, and p12E when compared to that of the wild-type virion. In contrast, two nonparalytogenic ts mutants, ts3 and ts10, like the wild-type virus, show normal processing of gPr80env in infected cells and production of virions with a similar amount of env proteins to that of the wild-type virion.
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PMID:A group of temperature-sensitive mutants of Moloney leukemia virus which is defective in cleavage of env precursor polypeptide in infected cells also induces hind-limb paralysis in newborn CFW/D mice. 683 18

Synchronized mouse cells (JLS-V9) chronically infected with Rauscher murine leukemia virus were used to study virus production, the synthesis of gag and env precursor proteins, and the expression of env protein on the cell surface during the cell cycle. The amount of virus released into the medium by synchronized cells during a 30-min interval was determined by using the XC plaque assay and by measuring reverse transcriptase activity. The results show that virus production occurs during mitosis. Labeling of the cell surface of synchronized cells with 125I or with fluorescein-conjugated antiserum shows that the amount of gp 70env on the cell surface parallels cellular growth. Therefore, the cell cycle-dependent release of virus is not accompanied by similar variations in the amount of viral envelope protein on the cell surface. Immunoprecipitation of cells labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was used to measure viral protein synthesis during the cell cycle. The rate of synthesis of gag precursor proteins show three maximums corresponding to the G1, middle S, and late S to G2 phases of the cell cycle. The rate of synthesis of env precursor proteins does not change, suggesting that in these cells the synthesis of these two gene products is controlled separately.
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PMID:Retrovirus gene expression during the cell cycle. I. Virus production, synthesis, and expression of viral proteins in Rauscher murine leukemia virus-infected mouse cells. 728 18

HTLV-II env protein, which cross-reacts with HTLV-I env, has been known to induce antibodies in infected individuals. In an attempt to suppress HTLV-II infection, we first generated HTLV-II env precursor glycoprotein (gp63) using baculovirus vector and the Sf-9 cell system. The production of antibodies to the cleaved env glycoprotein gp46 was verified by analysis of immunized rabbit sera as well as HTLV-II infected human sera using Western blotting. Moreover, the rabbit antibody was shown to suppress syncytial formation of the cells (Vines) infected with human T-cell leukemia virus type II (HTLV-II: HTLV-II-Vines). HTLV-II infection in rabbits was produced by intravenous injection of HTLV-II-Vines into female rabbits (New Zealand White). Antibody against HTLV-II could be detected by the 2nd week after inoculation, and its titer reached the maximum at the 10th week. Specific antibodies against env gp46 and gag p24 were detected in 2 of 2 rabbits and in 1 of 2 by Western blotting methods, respectively. Proviral DNA was detected by nested PCR, which was verified by Southern hybridization, at all times checked after inoculation, suggesting the persistence of infection, albeit at low levels. In the studies to determine if vaccination could protect against HTLV-II infection, rabbits were first immunized with the env protein, then were challenged by inoculation with HTLV-II-Vines as described above. Employing nested PCR, the provirus could not be detected at any time after challenge. The observation that active immunization could effectively protect rabbits from infection would seem to have important implications for equivalent vaccination of humans against both HTLV-I and HTLV-II.
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PMID:[Production of HTLV-II env protein and its suppressive effect on infection]. 759 Jun 8


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