Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have succeeded in growing the HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) in cultured human thymic epithelial (TE) cells. Expression of the HIV-1 proteins p17 and p24 was detected by immunofluorescence and reached a peak at 3 days after infection. Antigen capture and reverse transcriptase assays were used to detect HIV-1 in culture fluids, with positive results also being realized. The infection was cytolytic; cellular disarrangement, increased numbers of Hassall's corpuscles, and giant cells first appeared in monolayers of TE cells at 2 days after inoculation. By 4 days these changes were increased, and by 7 days, retraction and involution of TE cells were evident. The infection of TE cells by HIV-1 was blocked by preincubation with monoclonal OKT4A antibodies directed against CD4 target molecules.
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PMID:Infection of cultured human thymic epithelial cells by human immunodeficiency virus. 246 78

Infectious retrovirus particles consist of a core structure containing RNA and gag-pol polypeptides surrounded by a lipid membrane studded with env proteins. A recombinant vaccinia virus was designed to express the entire gag-pol precursor protein of the human immunodeficiency virus type 1. Synthesis and processing of gag proteins occurred in mammalian cells infected with this live recombinant virus, and reverse transcriptase was detected largely in the medium. Electron micrographs revealed immature retrovirus-like particles budding from the plasma membrane and extracellular particles with morphological characteristics of immature and mature human immunodeficiency virus. The latter contained functional reverse transcriptase as well as processed p24 and p17 gag polypeptides. Thus, assembly and maturation of human immunodeficiency virus-like particles can occur in the absence of either infectious RNA molecules or env proteins. The production of noninfectious virus-like particles by expression vectors should be useful for biochemical studies and could provide a safe source of material for the development of vaccines.
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PMID:Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector. 247 31

A family of oligonucleotides and phosphorothioate oligonucleotide analogues was synthesized with a cholesteryl group tethered at the 3'-terminal internucleoside link. This modification, introduced to enhance interaction of the polyanions with cell membranes, significantly increases the antiviral activity of the oligomers, as judged by inhibition of syncytia formation and expression of viral proteins p17, p24, and reverse transcriptase for human immunodeficiency virus 1 in Molt-3 cells. In the most favorable case, with a 20-mer cholesteryl-phosphorothioate derivative, complete inhibition by all assays was obtained with an oligomer concentration of 0.2 microM. Even decamers were active, and some antiviral activity was observed for a heptanucleotide cholesteryl-phosphorothioate derivative, which binds very poorly to complementary oligonucleotides. These facts, and the finding that the activity of the phosphorothioate decamers does not correlate with a specific sequence, suggests that a mechanism other than "antisense inhibition" may be operative in these systems.
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PMID:Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. 277 42

Over a period of six months, we have followed a total of six different EBV-transformed B cell lines, each of which has been infected by the human immunodeficiency virus (HIV-1). The results indicate that all of these lines were initially able to produce progeny HIV-1, but that over time three of them ceased to produce infectious virus and two lines failed to elaborate significant levels of reverse transcriptase activity into the medium. We further found that two of the latter cell lines continued to express the viral antigens p17, p24 and/or gp41, as determined by indirect immunofluorescence assay, at times after progeny HIV-1 could no longer be detected. Those cell lines which continued to secrete progeny HIV-1 did so intermittently with large amounts of virus production on some days but not others. We further found that treatment of such cells with 3' azido-3'-deoxythymidine (AZT) completely inhibited production of progeny HIV-1.
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PMID:Heterogeneity of HIV-1 replication and antigen expression in EBV-transformed B cell lines. 284

In order to facilitate the purification of recombinant proteins for immunization purposes, for example through the construction of solid matrix-antibody-antigen (SMAA) complexes, two small but different tag sequences were attached to the N- and C-termini of recombinant proteins. The 12-amino-acid N-terminal tag (His) contained an array of six histidines which permitted first-step purification by nickel-affinity column chromatography. The C-terminal tag (Pk) was a 14-amino-acid oligopeptide recognized by the monoclonal antibody (mAb) SV5-P-k. The mAb SV5-P-k was linked to a solid matrix and the solid matrix-antibody complexes were saturated with PK-linked recombinant antigens to generate SMAA complexes. The procedure used for construction of the SMAA complexes also acted as a second purification step. Neither of the tag sequences was cleaved from the recombinant proteins before immunization. This two-step purification procedure was used to construct SMAA complexes containing either p17 or reverse transcriptase (rt) of simian immunodeficiency virus (SIV). Mice immunized with these complexes had high antibody titres recognizing both the respective recombinant and native SIV proteins. A weak antibody response was also measured against both the terminal tags. The advantages of using simple dual purification procedures for isolating tag-linked recombinant proteins for use in vaccines are discussed.
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PMID:Two-tag purification of recombinant proteins for the construction of solid matrix-antibody-antigen (SMAA) complexes as vaccines. 750 59

In this study, we have characterized the HIV DNA-containing replication complexes present in cells early after cell-to-cell infection, using sucrose gradient sedimentation and immunoprecipitation. Six hours after cell-to-cell infection, a cytoplasmic HIV replication complex sedimented as a large structure (320S). This replication complex was precipitated by antisera to three virus-coded enzymes (reverse transcriptase, integrase, protease), to the matrix protein (p17), and to cellular histones but not to the major capsid protein (p24). This replication complex was not associated with cell membranes and could not be dissociated into smaller discrete subunits, using detergents. Nuclear extracts from the same cell-to-cell infection contained a smaller (80S) complex that lacked reverse transcriptase and matrix protein (p17). Cytoplasmic replication complexes from a cell-free virus infection sedimented as 160S structures under identical conditions, as previously reported. Our results indicate that, following cell-to-cell transmission of HIV, all the HIV pol gene products, the matrix protein p17, and cellular histones are present in cytoplasmic replication complexes that are taking part in or have completed reverse transcription. Transportation of the cytoplasmic replication complex to the nucleus is associated with structural changes, including a reduction in size and altered protein composition.
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PMID:Characterization of HIV replication complexes early after cell-to-cell infection. 750 34

For diagnosis of HIV-1 infection, attempts were made to detect anti-HIV-1 IgG in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT) and p17 as antigens. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate. The enzymes used as labels were horseradish peroxidase for RT and Escherichia coli beta-D-galactosidase for p17. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, bound enzyme activity was assayed by fluorometry. Urine samples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positivity was confirmed by preincubation of urine samples with excess of the antigens. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine samples. Detection of anti-HIV-1 IgG in urine by the immune complex transfer enzyme immunoassay using different antigens would make diagnosis of HIV-1 infection possible.
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PMID:Detection of antibody IgG to HIV-1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV-1 infection. 750 5

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p17 and p24 as antigens, and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-beta-D-galactosidase conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of epsilon N-2,4-dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 100%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p17 and p24 as antigens. The sensitivity could be improved by a longer assay of bound beta-D-galactosidase activity by using concentrated urine samples and by the combined use of recombinant RT, p17, and p24. Thus, reliable diagnosis of HIV-1 infection was possible for asymptomatic carriers.
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PMID:Diagnosis of HIV-1 infection by detection of antibody IgG to HIV-1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens. 752 37

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.
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PMID:Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. 768 10

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
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PMID:Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses. 807 30


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