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: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52

Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.
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PMID:In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. 138 71

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent trans-activator of the viral long terminal repeat (LTR). The N-terminal region of Tat is rich in proline and acidic residues analogous to the activation domains of other transcription factors such as GAL-4 and CTF/NF-1. Several basic residues are also present in this region. To investigate the role of these structural features in the Tat-mediated trans-activation, we have chemically synthesized and evaluated Tat analogs with alanine or glutamine replacing one or more of these amino acid residues. Our data show that substitution of Glu-2, His-13, or all the proline in the Pro-Xaa3-Pro triad drastically reduced activity. In contrast, changes at Arg-7, Lys-12 and any one proline residue in the triad moderately reduced, and substitution of Lys-19 showed little effect on, activity. These results show that the native structure of the N-terminal 19 amino acid sequence is essential for Tat function, and that the overall topology of this domain and not the acidic residues alone appears necessary for trans-activation.
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PMID:Functional analysis of the N-terminal domain of Tat protein of the human immunodeficiency virus type 1. 150 86

Two immunologically distinct glycoproteins, fractions C4 and C6, with a molecular weight of 28,000 and 28,500, respectively, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were isolated from seeds of Luffa cyclindrica using acetone precipitation, gel filtration on Sephadex G-75, and ion exchange chromatography on CM-Sepharose CL-6B. Fractions C4 and C6 correspond to luffin-a and luffin-b, respectively, according to the ion exchange chromatographic behavior and amino acid compositions. Fraction C6 and luffin-b were characterized by a lower content of threonine and a higher content of proline than fraction C4 and luffin-a. The 2 luffins, the protein from Luffa acutangula (luffaculin) and trichosanthin exhibited an overall similarity in amino acid composition. The proteins differed in the content of aspartic acid, threonine, proline, and alanine but were otherwise similar in amino acid composition. The ribosome inactivating proteins from Luffa cylindrica seeds also possessed abortifacient activity: they were capable of inducing mid term abortion in mice, inhibiting protein synthesis in a cell-free system, and suppressing thymidine uptake by human choriocarcinoma cells. The abortifacient activity of these proteins is possibly the result of their inhibitory effects on the biosynthetic activity of implanting embryos and endometrial cells. Trichosanthin inhibits the replication of human immunodeficiency virus (HIV) in acutely and chronically infected cells of lymphocyte and mononuclear phagocyte lineage with a potential in AIDS therapy. However, it is still unknown whether the proteins from Luffa cylindrica seeds also possess anti-HIV activity.
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PMID:Two proteins with ribosome-inactivating, cytotoxic and abortifacient activities from seeds of Luffa cylindrica roem (Cucurbitaceae). 150 59

The synthesis of several novel carbocyclic purine nucleosides that incorporate a nitrogen in place of carbon 3 of the cyclopentyl moiety are described. These analogues are all derived from the key stereochemically defined intermediate N-(tert-butoxycarbonyl)-O-[(4-methoxyphenyl)diphenylmethyl]-trans- 4- hydroxy-D-prolinol (19), which was accessible in 61.1% overall yield for a five-step sequence starting from cis-4-hydroxy-D-proline. The heterocyclic bases, 6-chloropurine and 2-amino-6-chloropurine, are efficiently introduced onto the pyrrolidine ring via a Mitsunobu-type coupling procedure with triphenylphosphine and diethyl azodicarboxylate. Standard transformations and removal of protecting groups gave the cis-adenine (26), hypoxanthine (27), 2,6-diaminopurine (28), and guanine (29) D-prolinol derivatives. In addition, a related sequence from trans-4-hydroxy-L-proline provided the enantiomeric L-prolinol guanine derivative (36). Lastly, the 6-(dimethylamino)purine analogue, 37, was coupled to N-(benzyl-oxycarbonyl)-p-methoxy-L-phenylalanine to provide, after deprotection, the novel puromycin-like analogue 39. The analogues 26-29, 36, and 39 were all evaluated for antitumor and, except for 39, for antiviral activity. These compounds failed to appreciably inhibit the growth of P388 mouse leukemia cells in vitro at concentrations up to 100 micrograms/mL. In addition, they did not exhibit noticeable activity against the human immunodeficiency virus or herpes simplex virus type 1 at concentrations as high as 100 microM. The adenine analogue, 26, did, however, prove to be a substrate for adenosine deaminase. It possessed an affinity for the enzyme only 50% less than that of adenosine with a Ki = 85 microM.
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PMID:Synthesis and biological evaluation of 4-purinylpyrrolidine nucleosides. 165 29

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
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PMID:Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). 168 23

The V3 loop (residues 303-338) of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope protein represents a principal neutralizing determinant for the virus. An HIV-1 proviral clone containing a mutation in the V3 loop was constructed in which the proline residue at position 313 was changed to an alanine (P313-A). This mutation alters the conserved GPGR sequence that is found in the V3 loop sequences of different HIV-1 isolates. The P313-A clone produced virus particles, which were infectious for a number of T-cell lines including MOLT-4, CEM, and SupT1, but demonstrated a relatively low infectivity on the AA5 B-cell line when compared with wild-type viruses, HTLV-IIIB, HXB2/10 (a chimeric molecular clone), and another mutant virus (Q290-T). V3 loop-specific neutralizing polyclonal sera and the 9284 monoclonal antibody, which recognizes the amino side of the V3 loop sequence, effectively blocked infectivity and syncytia formation of all viruses tested. In contrast, the 0.5 beta monoclonal antibody, which is biologically more potent than 9284 and recognizes a different V3 loop determinant, failed to neutralize the P313-A virus. These results suggest that the proline residue in the relatively conserved GPGR "turn" region of the V3 loop is crucial for recognition by the 0.5 beta antibody. The observed variation in sensitivity of the B-cell line to the P313-A virus may reflect the presence of cell-specific factors which could be important in establishing an HIV-1 infection.
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PMID:Alteration of HIV-1 infectivity and neutralization by a single amino acid replacement in the V3 loop domain. 176 61

The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.
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PMID:First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form. 195 5

The virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.
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PMID:Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. 198 79

We have isolated a variant of human immunodeficiency virus type 1 (HIV-1) which is highly infectious to fibroblastlike cells (BT cells) derived from human brain as well as CD4-positive T cells. This variant HIV-1, named HIV[GUN-1V], was obtained by infecting BT cells with a prototype HIV-1 isolate, named HIV[GUN-1WT], which is highly infectious to T cells but barely infectious to BT cells. HIV[GUN-1V] infects BT cells productively and this infection appeared to be mediated by CD4. To elucidate the viral gene responsible for the host range difference between the variant and prototype HIV-1s, we cloned and analyzed the provirus genomes of the two viruses. Examination of the infectivities of BT cells by various recombinant viruses and analyses of the nucleotide sequences of HIV[GUN-1V] and HIV[GUN-1WT] showed that a single nucleotide exchange was responsible for their difference in infectivity of BT cells: HIV[GUN-1V] contains a thymine residue instead of the cytosine residue in HIV[GUN-1WT] at position 931 of the env coding sequence. Replacement of cytosine by thymine at this position of the env coding sequence of the HIV[GUN-1WT] genome induced the ability to infect BT cells. The base exchange at this position was expected to change amino acid 311 of the envelope glycoprotein, gp120, from proline to serine, which is located in a variable region containing type-specific immunodominant epitopes. Thus, HIV[GUN-1V] acquired a wider host range than HIV[GUN-1WT] by a single point mutation in the env gene.
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PMID:Host range mutant of human immunodeficiency virus type 1: modification of cell tropism by a single point mutation at the neutralization epitope in the env gene. 200 39


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