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1.
The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR. 相似文献
2.
Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in
anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved
pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to
be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected
for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine
substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate
envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate
complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease. 相似文献
3.
Relative fitness and replication capacity of a multinucleoside analogue-resistant clinical human immunodeficiency virus type 1 isolate with a deletion of codon 69 in the reverse transcriptase coding region
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Villena C Prado JG Puertas MC Martínez MA Clotet B Ruiz L Parkin NT Menéndez-Arias L Martinez-Picado J 《Journal of virology》2007,81(9):4713-4721
4.
Zhigang Liu Yong Wang Ravikiran S. Yedidi Tamaria G. Dewdney Samuel J. Reiter Joseph S. Brunzelle Iulia A. Kovari Ladislau C. Kovari 《Biochemical and biophysical research communications》2013,430(3):1022-1027
The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease. 相似文献
5.
Hiroyuki Gatanaga Yasuhiro Suzuki Hsinyi Tsang Kazuhisa Yoshimura Mark F Kavlick Kunio Nagashima Robert J Gorelick Sek Mardy Chun Tang Michael F Summers Hiroaki Mitsuya 《The Journal of biological chemistry》2002,277(8):5952-5961
Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1. 相似文献
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Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.
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Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule. 相似文献
8.
Benjamin D. Kuiper Bradley J. Keusch Tamaria G. Dewdney Poorvi Chordia Kyla Ross Joseph S. Brunzelle Iulia A. Kovari Rodger MacArthur Hossein Salimnia Ladislau C. Kovari 《Biochemistry and Biophysics Reports》2015
HIV-1 protease (PR) is a 99 amino acid protein responsible for proteolytic processing of the viral polyprotein – an essential step in the HIV-1 life cycle. Drug resistance mutations in PR that are selected during antiretroviral therapy lead to reduced efficacy of protease inhibitors (PI) including darunavir (DRV). To identify the structural mechanisms associated with the DRV resistance mutation L33F, we performed X-ray crystallographic studies with a multi-drug resistant HIV-1 protease isolate that contains the L33F mutation (MDR769 L33F). In contrast to other PR L33F DRV complexes, the structure of MDR769 L33F complexed with DRV reported here displays the protease flaps in an open conformation. The L33F mutation increases noncovalent interactions in the hydrophobic pocket of the PR compared to the wild-type (WT) structure. As a result, L33F appears to act as a molecular anchor, reducing the flexibility of the 30s loop (residues 29–35) and the 80s loop (residues 79–84). Molecular anchoring of the 30s and 80s loops leaves an open S1/S1′ subsite and distorts the conserved hydrogen-bonding network of DRV. These findings are consistent with previous reports despite structural differences with regards to flap conformation. 相似文献
9.
The conserved carboxy terminus of the capsid domain of human immunodeficiency virus type 1 gag protein is important for virion assembly and release 总被引:3,自引:0,他引:3
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Melamed D Mark-Danieli M Kenan-Eichler M Kraus O Castiel A Laham N Pupko T Glaser F Ben-Tal N Bacharach E 《Journal of virology》2004,78(18):9675-9688
The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an alpha-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process. 相似文献
10.
Yong Wang Zhigang Liu Joseph S. Brunzelle Iulia A. Kovari Tamaria G. Dewdney Samuel J. Reiter Ladislau C. Kovari 《Biochemical and biophysical research communications》2011,(4):737
Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82 mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants. 相似文献
11.
Selective escape from CD8+ T-cell responses represents a major driving force of human immunodeficiency virus type 1 (HIV-1) sequence diversity and reveals constraints on HIV-1 evolution 总被引:6,自引:0,他引:6
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Allen TM Altfeld M Geer SC Kalife ET Moore C O'sullivan KM Desouza I Feeney ME Eldridge RL Maier EL Kaufmann DE Lahaie MP Reyor L Tanzi G Johnston MN Brander C Draenert R Rockstroh JK Jessen H Rosenberg ES Mallal SA Walker BD 《Journal of virology》2005,79(21):13239-13249
The sequence diversity of human immunodeficiency virus type 1 (HIV-1) represents a major obstacle to the development of an effective vaccine, yet the forces impacting the evolution of this pathogen remain unclear. To address this issue we assessed the relationship between genome-wide viral evolution and adaptive CD8+ T-cell responses in four clade B virus-infected patients studied longitudinally for as long as 5 years after acute infection. Of the 98 amino acid mutations identified in nonenvelope antigens, 53% were associated with detectable CD8+ T-cell responses, indicative of positive selective immune pressures. An additional 18% of amino acid mutations represented substitutions toward common clade B consensus sequence residues, nine of which were strongly associated with HLA class I alleles not expressed by the subjects and thus indicative of reversions of transmitted CD8 escape mutations. Thus, nearly two-thirds of all mutations were attributable to CD8+ T-cell selective pressures. A closer examination of CD8 escape mutations in additional persons with chronic disease indicated that not only did immune pressures frequently result in selection of identical amino acid substitutions in mutating epitopes, but mutating residues also correlated with highly polymorphic sites in both clade B and C viruses. These data indicate a dominant role for cellular immune selective pressures in driving both individual and global HIV-1 evolution. The stereotypic nature of acquired mutations provides support for biochemical constraints limiting HIV-1 evolution and for the impact of CD8 escape mutations on viral fitness. 相似文献
12.
Domains of the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions. 总被引:20,自引:18,他引:2
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We recently demonstrated that a single amino acid substitution in matrix residue 12 (12LE) or 30 (30LE) blocks the incorporation of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into virions and that this block can be reversed by pseudotyping with heterologous retroviral envelope glycoproteins with short cytoplasmic tails or by truncating the cytoplasmic tail of HIV-1 transmembrane glycoprotein gp41 by 104 or 144 amino acids. In this study, we mapped the domain of the gp41 cytoplasmic tail responsible for the block to incorporation into virions by introducing a series of eight truncation mutations that eliminated 23 to 93 amino acids from the C terminus of gp41. We found that incorporation into virions of a HIV-1 envelope glycoprotein with a deletion of 23, 30, 51, or 56 residues from the C terminus of gp41 is specifically blocked by the 12LE matrix mutation, whereas truncations of greater than 93 amino acids reverse this defect. To elucidate the role of matrix residue 12 in this process, we introduced a number of additional single amino acid substitutions at matrix positions 12 and 13. Charged substitutions at residue 12 blocked envelope incorporation and virus infectivity, whereas more subtle amino acid substitutions resulted in a spectrum of envelope incorporation defects. To characterize further the role of matrix in envelope incorporation into virions, we obtained and analyzed second-site revertants to two different matrix residue 12 mutations. A Val-->Ile substition at matrix amino acid 34 compensated for the effects of both amino acid 12 mutations, suggesting that matrix residues 12 and 34 interact during the incorporation of HIV-1 envelope glycoproteins into nascent virions. 相似文献
13.
Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity
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Logsdon BC Vickrey JF Martin P Proteasa G Koepke JI Terlecky SR Wawrzak Z Winters MA Merigan TC Kovari LC 《Journal of virology》2004,78(6):3123-3132
The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (A) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease "flaps" stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 A. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k(off) rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k(on) and k(off) data (K(d) = k(off)/k(on)) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms. 相似文献
14.
Exploring Nonnatural Evolutionary Pathways by Saturation Mutagenesis: Rapid Improvement of Protein Function 总被引:1,自引:0,他引:1
Random point mutagenesis does not access a large fraction of protein sequence space corresponding to primarily nonconservative
amino acid substitutions. The cost of this limitation during directed evolution is unknown. Random point mutagenesis over
the entire gene encoding the psychrophilic protease subtilisin S41 identified a pair of residues (Lys211 and Arg212) where
mutations provided significant increases in thermostability. These were subjected to saturation mutagenesis to test whether
the amino acids not easily accessible by point mutagenesis provide even better ``solutions' to the thermostabilization challenge.
A significant fraction of these variants surpassed the stability of the variants with point mutations. DNA sequencing revealed
highly hydrophobic residues in the four most stable variants (Pro/Ala, Pro/Val, Leu/Val, and Trp/Ser). These nonconservative
replacements, accessible only by multiple (two to three) base substitutions in a single codon, would be extremely rare in
a point mutation library. Such replacements are also extremely rare in natural evolution. Saturation mutagenesis may be used
advantageously during directed evolution to explore nonnatural evolution pathways and enable rapid improvement in protein
traits.
Received: 15 March 1999 / Accepted: 28 June 1999 相似文献
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Comprehensive bioinformatic analysis of the specificity of human immunodeficiency virus type 1 protease
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Rapidly developing viral resistance to licensed human immunodeficiency virus type 1 (HIV-1) protease inhibitors is an increasing problem in the treatment of HIV-infected individuals and AIDS patients. A rational design of more effective protease inhibitors and discovery of potential biological substrates for the HIV-1 protease require accurate models for protease cleavage specificity. In this study, several popular bioinformatic machine learning methods, including support vector machines and artificial neural networks, were used to analyze the specificity of the HIV-1 protease. A new, extensive data set (746 peptides that have been experimentally tested for cleavage by the HIV-1 protease) was compiled, and the data were used to construct different classifiers that predicted whether the protease would cleave a given peptide substrate or not. The best predictor was a nonlinear predictor using two physicochemical parameters (hydrophobicity, or alternatively polarity, and size) for the amino acids, indicating that these properties are the key features recognized by the HIV-1 protease. The present in silico study provides new and important insights into the workings of the HIV-1 protease at the molecular level, supporting the recent hypothesis that the protease primarily recognizes a conformation rather than a specific amino acid sequence. Furthermore, we demonstrate that the presence of 1 to 2 lysine residues near the cleavage site of octameric peptide substrates seems to prevent cleavage efficiently, suggesting that this positively charged amino acid plays an important role in hindering the activity of the HIV-1 protease. 相似文献
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