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)

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

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.
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PMID:DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein. 805 66

The interaction of human immunodeficiency virus (HIV) nucleocapsid protein (NCp) with a substrate closely mimicking a retrovirus replication intermediate was studied. The heteroduplex substrate consisted of a DNA and RNA of 80 and 63 nucleotides, respectively. The nucleotides at the 3' end of the DNA were complementary to those at the 3' end of the RNA such that a hybrid region of 30 base pairs could form. HIV-reverse transcriptase (RT) extended the DNA and cleaved the RNA strand of the substrate. The rates of extension and cleavage were significantly decreased when the substrate was prebound with NCp before HIV-RT addition. In assays assessing the integrity of the substrate by measuring release of the DNA strand from the heteroduplex, prebinding with NCp protected the substrate when HIV-RT was added, a result consistent with resistance to RT-mediated cleavage. In contrast, NCp significantly decreased the thermal stability of the substrate as judged by incubation of the substrate at various temperatures. In strand transfer assays, a 189-nucleotide RNA (acceptor) with an internal region complementary to all 80 nucleotides of the substrate DNA was incubated with the substrate in the presence or absence of NCp. Nucleocapsid protein stimulated strand transfer in which the substrate RNA was displaced upon binding of the DNA to the acceptor. Results are discussed with respect to the role of NCp in retroviral recombination.
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PMID:Interaction of human immunodeficiency virus nucleocapsid protein with a structure mimicking a replication intermediate. Effects on stability, reverse transcriptase binding, and strand transfer. 866 19

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.
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PMID:NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer. 896 39

Nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein containing two zinc fingers of the form (CX2CX4HX4C) and is present at about 2,000 copies inside the viral core. NCp7 molecules are tightly associated with the genomic RNA dimer to form the nucleocapsid, which also includes reverse transcriptase and integrase proteins. In vitro, NCp7 has been shown to bind specifically to HIV-1 RNA, inducing NCp7-NCp7 interactions. In the viral context, mutagenesis of amino acid residues in the zinc finger domains showed that NCp7 is responsible for the specific incorporation of genomic RNA into virions and is necessary for correct virion assembly and maturation. In this work, we investigated the consequences of mutating conserved basic residues in the N-terminal region that precedes the first zinc finger. Two of the mutants were poorly infectious and showed only limited, though significant, defects in RNA encapsidation and viral protein maturation. Electron microscopy, together with sucrose gradient analysis, revealed defects in particle core structure and heterogeneity among mutant virions. These defects were associated with strong reduction of proviral DNA synthesis and stability in newly infected cells. Taken together, these data show multiple and probably interdependent implications for the NCp7 protein in both early and late phases of the HIV-1 replicative cycle and emphasize it as a target for antiviral drug development.
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PMID:Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis. 926 26

A system to study the fidelity of internal strand transfer events was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. The homology occurred over a 119-base internal region of the donor which coded for the N-terminal portion of the alpha-lac gene. Polymerase chain reaction (PCR) was used to amplify DNA synthesis products. The PCR products were then digested with PvuII and EcoRI and ligated into a vector which had this same region excised. Transformed Escherichia coli were screened for the ability to produce a functional beta-galactosidase protein by blue-white phenotype analysis with white colonies scored as those with errors in alpha-lac. Products synthesized on the donor were used to assess the error rate of human immunodeficiency virus-RT while products transferring to and subsequently extended on the acceptor (transfer products) were used to monitor transfer fidelity. Human immunodeficiency virus-RT made approximately 1 error per 7500 bases copied in the assay. Nucleocapsid protein (NCp), although stimulating strand transfer 3-fold, had no effect on RT fidelity. Transfer products in the absence of NCp had essentially the same amount of errors as donor-directed products while those produced with NCp showed a slight increase in error frequency. Overall, strand transfer events on this template were highly accurate. Since experiments with other templates have suggested that transfer is error prone, the fidelity of strand transfer may be highly sequence dependent.
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PMID:High fidelity of internal strand transfer catalyzed by human immunodeficiency virus reverse transcriptase. 943 Jun 86

Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4CX4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.
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PMID:Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication. 955 38

Nucleocapsid protein (NCp7), which contains highly conserved retroviral zinc fingers, is essential in the early as well as the late phase of human immunodeficiency virus (HIV) life cycle and constitutes a novel target for AIDS therapy. HIV-1 NCp7 is a basic 55 amino acid protein containing two C(X)2C(X)4H(X)4C motif zinc fingers flanked by basic amino acids on each side. 2,2'-dithiobisbenzamides have previously been reported to release zinc from these NCp7 zinc fingers and also to inhibit HIV replication. Specifically, 2,2'-dithiobisbenzamides derived from simple amino acids showed good antiviral activities. The benzisothiazolone 3, the cyclic derivative of 2, was selected for clinical trials as an agent for AIDS therapy. Herein we report the syntheses and antiviral activities, including therapeutic indices, of 2,2'-dithiobisbenzamides derived from alpha-, beta- and gamma-amino acids. Electrospray ionization mass spectrometry was used to study the zinc-ejection activity of these compounds. Among the alpha-amino acid derived 2,2'-dithiobisbenzamides, analogues containing alkyl side chains were found to be antivirally active with good therapeutic indices. 2,2'-Dithiobisbenzamides, derived from beta- and gamma-amino acids, were found to possess better antiviral and therapeutic efficacies than the alpha-amino acid analogues. Thus compound 59 was found to possess an EC50 of 1.9 microM with a therapeutic index of > 50. Interestingly, 2,2'-dithiobisbenzamides derived from alpha-amino acids containing a protected acid function and polar side chains also exhibited very good antiviral activity.
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PMID:2,2'-Dithiobisbenzamides derived from alpha-, beta- and gamma-amino acids possessing anti-HIV activities: synthesis and structure-activity relationship. 983 2

The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism.
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PMID:Template dimerization promotes an acceptor invasion-induced transfer mechanism during human immunodeficiency virus type 1 minus-strand synthesis. 1266 78

Template switching during reverse transcription contributes to recombination in human immunodeficiency virus type 1 (HIV-1). Our recent studies suggest that the process can occur through a multi-step mechanism involving RNase H cleavage, acceptor invasion, branch migration, and finally primer terminus transfer. In this study, we analyzed the effects of reverse transcriptase (RT)-pausing, RNase H cleavages and template structure on the transfer process. We designed a series of donor and acceptor template pairs with either minimal pause sites or with pause sites at various locations along the template. Restriction sites within the region of homology allowed efficient mapping of the location of primer terminus transfer. Blocking oligomers were used to probe the acceptor invasion site. Introduction of strong pause sites in the donor increased transfer efficiency. However, the new pauses were not necessarily associated with effective invasion. In this system, the primary invasion occurred at a region of donor cleavage associated with weak pausing. These results together with acceptor structure predictions indicated that a potential invasion site is used only in conjunction with a favorable acceptor structure. Stabilizing acceptor structure at the predicted invasion region lowered the transfer efficiency, supporting this conclusion. Differing from previous studies, terminus transfer occurred at a short distance from the invasion site. Introduction of structure into the acceptor template shifted the location of terminus transfer. Nucleocapsid protein, which can improve cDNA-acceptor interactions, increased transfer efficiency with some shift of terminus transfer closer to the invasion site. Overall results support that the acceptor structure has a major influence on the efficiency and position of the invasion and terminus transfer steps.
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PMID:Effects of donor and acceptor RNA structures on the mechanism of strand transfer by HIV-1 reverse transcriptase. 1621 74


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