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 reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571-3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.
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PMID:HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro. 1007 49

Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.
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PMID:The three-dimensional structure of a Tn5 transposase-related protein determined to 2.9-A resolution. 1020 11

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.
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PMID:X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform. 1066 6

Integration of retrovirus DNA is a specific process catalyzed by the integrase protein acting to join the viral substrate DNA (att) sequences of about 10 bases at the ends of the long terminal repeat (LTR) to various sites in the host target cell DNA. Although the interaction is sequence specific, the att sequences of different retroviruses are largely unrelated to one another and usually differ between the two ends of the viral DNA. To define substrate sequence specificity, we designed an "in vitro evolution" scheme to select an optimal substrate sequence by competitive integration in vitro from a large pool of partially randomized substrates. Integrated substrates are enriched by PCR amplification and then regenerated and subjected to subsequent cycles of selection and enrichment. Using this approach, we obtained the optimal substrate sequence of 5'-ACGACAACA-3' for avian sarcoma-leukosis virus (ASLV) and 5'-AACA(A/C)AGCA-3' for human immunodeficiency virus type 1, which differed from those found at both ends of the viral DNA. Clonal analysis of the integration products showed that ASLV integrase can use a wide variety of substrate sequences in vitro, although the consensus sequence was identical to the selected sequence. By a competition assay, the selected nucleotide at position 4 improved the in vitro integration efficiency over that of the wild-type sequence. Viral mutants bearing the optimal sequence replicated at wild-type levels, with the exception of some mutations disrupting the U5 RNA secondary structure important for reverse transcription, which were significantly impaired. Thus, maximizing the efficiency of integration may not be of major importance for efficient retrovirus replication.
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PMID:Substrate sequence selection by retroviral integrase. 1115 9

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994-3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.
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PMID:Changes in the mechanism of DNA integration in vitro induced by base substitutions in the HIV-1 U5 and U3 terminal sequences. 1178 85

Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and Escherichia coli RNase H possess a positively charged alpha-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (DeltaC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5' and 3' viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5' end.
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PMID:Mutations of the RNase H C helix of the Moloney murine leukemia virus reverse transcriptase reveal defects in polypurine tract recognition. 1213 40

The vif gene, one of the six auxiliary genes of human immunodeficiency virus (HIV), is essential for virus propagation in peripheral blood lymphocytes and macrophages and in certain T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV type 1 (HIV-1) Gag-Pol polyproteins expressed in bacterial cells, as well as the protease-mediated cleavage of synthetic peptides in vitro. Peptides derived from the aa 78-98 region in the Vif molecule specifically inhibit and bind the HIV-1 protease in vitro and arrest the production of infectious viruses in HIV-1-infected cells. This study demonstrates that (i) purified recombinant Vif protein and HIV-1 but not avian sarcoma leukaemia virus protease specifically bind each other and (ii) the interaction between these two proteins takes place at the N terminus of the protease (aa 1-9) and the central part of Vif (aa 78-98). The data presented in this report suggest a model in which Vif interacts with the dimerization sites of the viral protease.
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PMID:Human immunodeficiency virus type 1 Vif binds the viral protease by interaction with its N-terminal region. 1218 77

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.
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PMID:Functional oligomeric state of avian sarcoma virus integrase. 1244 21

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.
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PMID:Characterization of a naphthalene derivative inhibitor of retroviral integrases. 1501

Recent studies have demonstrated that avian sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.
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PMID:Transduction of terminally differentiated neurons by avian sarcoma virus. 1507 71


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