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E Arnold

Publications and source records attributed to E Arnold.

At least 37 records · Page 2Linked to original sources
PubMed2001

HIV-1 reverse transcriptase mutations found in a drug-experienced patient confer reduced susceptibility to multiple nucleoside reverse transcriptase inhibitors.

HIV-1 reverse transcriptase (RT) genotypes were obtained from 13 patients treated with stavudine. No previously-reported mutations indicative of stavudine resistance were found in these patients and no novel mutations occurred in more than two patients. One patient, treated with stavudine for 1 month and treated previously with zidovudine, zalcitabine and lamivudine, carried a mutation at codon 75 of the RT (V75M). A chimeric virus, including the patient's RT sequence from codon 25 to codon 220, which carried the resistance mutations M41 L, D67N, T69D, K70R, L210W and T215Y in addition to V75M, displayed reduced susceptibility to multiple nucleoside RT inhibitors (NRTIs). Removal of V75M from this RT background resulted in a return of susceptibility to didanosine and lamivudine. Our data are in agreement with previous studies demonstrating the rarity of stavudine resistance mutations in stavudine-treated patients. However, we describe a new set of mutations, found in the RT of a heavily-treated patient, that can confer reduced susceptibility to multiple NRTIs. These results underscore the importance of increased vigilance for possible multiple-drug resistance in patients who have been heavily treated with NRTIs.

Acquired Immunodeficiency Syndrome↗
PubMed2000

The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase.

Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.

Amino Acid Substitution↗
PubMed2000

Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase.

We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116-Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.

Binding Sites↗
PubMed2000

Mind over matter.

Explore the source record for details and available documents.

Acute Disease↗
PubMed2000

A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir.

Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12. 5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy.

Anti-HIV Agents↗
PubMed1999

Similarities and differences in the RNase H activities of human immunodeficiency virus type 1 reverse transcriptase and Moloney murine leukemia virus reverse transcriptase.

Retroviral revXerse transcriptases (RTs) have an associated RNase H activity that can cleave RNA-DNA duplexes with considerable precision. We believe that the structure of the RNA-DNA duplexes in the context of RT determines the specificity of RNase H cleavage. To test this idea, we treated three related groups of synthetic RNA-DNA hybrids with either Moloney murine leukemia virus (MLV) RT or human immunodeficiency virus type 1 (HIV-1) RT. All of the hybrids were prepared using the same 81-base RNA template. The first series of RNase H substrates was prepared with complementary DNA oligonucleotides of different lengths, ranging from 6 to 20 nucleotides, all of which shared a common 5' end and were successively shorter at their 3' ends. The second series of oligonucleotides had a common 3' end but shorter 5' ends. The DNA oligonucleotides in the third series were all 20 bases long but had non-complementary stretches at either the 5' end, 3' end, or both ends. Several themes have emerged from the experiments with these RNA-DNA duplexes. (1) Both HIV-1 RT and MLV RT cleave fairly efficiently if the duplex region is at least eight bases long, but not if it is shorter. (2) Although, under the conditions we have used, both enzymes require the substrate to have a region of RNA-DNA duplex, both MLV RT and HIV-1 RT can cleave RNA outside the region that is part of the RNA-DNA duplex. (3) The polymerase domain of HIV-1 RT uses certain mismatched segments of RNA-DNA to position the enzyme for RNase H cleavage, whereas the polymerase domain of MLV RT does not use the same mismatched segments to define the position for RNase H cleavage. (4) For HIV-1 RT, a mismatched region near the RNase H domain can interfere with RNase H cleavage; cleavage is usually (but not always) more efficient if the mismatched segment is deleted. These results are discussed in regard to the structure of HIV-1 RT and the differences between HIV-1 RT and MLV RT.

Amino Acid Sequence↗
PubMed1999

Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids.

An important component of triple-drug anti-AIDS therapy is 2', 3'-dideoxy-3'-thiacytidine (3TC, lamivudine). Single mutations at residue 184 of the reverse transcriptase (RT) in HIV cause high-level resistance to 3TC and contribute to the failure of anti-AIDS combination therapy. We have determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant structures are very similar. However, comparison of crystal structures of M184I mutant and wild-type HIV-1 RT with and without DNA reveals repositioning of the template-primer in the M184I/DNA binary complex and other smaller changes in residues in the dNTP-binding site. On the basis of these structural results, we developed a model that explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analog 3TCTP. In this model, steric hindrance is expected for NRTIs with beta- or L- ring configurations, as with the enantiomer of 3TC that is used in therapy. Steric conflict between the oxathiolane ring of 3TCTP and the side chain of beta-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. The model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants. Repositioning of the template-primer as observed in the binary complex (M184I/DNA) may also occur in the catalytic ternary complex (M184I/DNA/3TCTP) and contribute to 3TC resistance by interfering with the formation of a catalytically competent closed complex.

Allosteric Regulation↗
PubMed1999

Major subdomain rearrangement in HIV-1 reverse transcriptase simulated by molecular dynamics.

We have performed eight 1-ns in vacuo molecular dynamics simulations of HIV-1 reverse transcriptase (RT). Starting with the p66 thumb subdomain in an upright configuration, the p66 thumb moved down over the palm during six of the eight trajectories, in excellent agreement with the crystallographic structure of unliganded RT. The large rearrangement of the p66 thumb subdomain, its tip moving approximately 30 A, occurs during the first 30-200 ps. This approach may allow a detailed study of the processes involved in biologically significant conformational changes in macromolecules.

Crystallography, X-Ray↗
PubMed1999

Pivot residue: an analysis of domain motion in proteins.

In this study, we present an approach to identify some residues that represent the pivot points to experience conformational changes between open (unligand) and closed (ligand) forms of a protein. First, an angle, theta, formed by 4 consecutive Ca atoms in polypeptide backbones was introduced. The difference of this angle, deltatheta, from the equivalent residues between the open and the closed form was used to represent the local torsion changes in the protein structure, and the residue with the maximum among deltatheta was identified to be a pivot residue. We demonstrate the ability of our method by identifying the pivot residues from five proteins, Lysozyme mutates, Lactoferrin, Lay/Arg/Orn-binding protein, Calmodulin and Catabolit gene activator protein. These pivot residues are located at the hinges in the proteins, they are hinge points for the domain motion. These examples also show that the pivot residues are useful to distinguish the mechanism between shear motion and hinge motion in a protein.

Amino Acid Sequence↗
PubMed1999

Alcohol consumption and psychological distress in recently widowed older men.

OBJECTIVE: The relationship between severe adverse life events and subsequent health-related behaviour is not well established for older people. To address this issue, we studied self-reported alcohol consumption in recently widowed older men and matched married men. METHOD: We carried out a double cohort study in community-residing men aged 65 years and over. Recently widowed men (n = 57; mean age = 74.5 years) were identified from the death records of their wives and assessed at 6 weeks, 6 months and 13 months post-bereavement. Matched married men (n = 57; mean age = 75.4 years) were identified from the electoral roll and assessed at similar intervals. Self-report measures of alcohol consumption, grief and state anxiety were employed. RESULTS: Similar proportions of older widowers and married men reported that they consumed some alcohol, although recently widowed older men reported significantly greater frequency (chi2 = 4.64, df = 1, p = 0.031) and quantity (chi2 = 7.75, df = 1, p = 0.005) of alcohol consumption than matched married men. A significant minority of subjects reported hazardous levels of alcohol consumption with 18.9% of widowers and 8.3% of married men reporting that they drank five or more standard drinks per drinking day. However, among widowers, self-reported alcohol consumption was not significantly correlated with levels of either self-reported grief or state anxiety. CONCLUSIONS: Hazardous alcohol consumption occurs commonly among recently widowed older men, but is not related to their levels of self-reported grief or psychological distress. Loss of spousal care and control may be an alternative explanation for this drinking behaviour. Older widowers should be regarded as a high-risk group for hazardous alcohol consumption and would be a suitable target group for preventive interventions.

Aged↗
PubMed1999

A disulfide-bound HIV-1 V3 loop sequence on the surface of human rhinovirus 14 induces neutralizing responses against HIV-1.

An immunogenic sequence from the V3 loop of the MN isolate of human immunodeficiency virus type 1 (HIV-1), His-Ile-Gly-Pro-Gly-Arg-Ala-Phe, was transplanted onto a surface loop of the VP2 capsid protein of human rhinovirus 14. To optimize for virus viability and immunogenicity of the transplanted sequence, the HIV sequence was flanked by (1) a cysteine residue that could form a disulfide bond and (2) randomized amino acids (in either of two arrangements) to generate numerous presentations of the Cys-Cys loop. The location for engineering in VP2 was chosen by searching the geometries of disulfide-bound loops in known protein structures. A model for the structure of the transplanted V3 loop sequence was developed using molecular dynamics and energy minimization calculations. Proteolytic digestion with and without reducing agent demonstrated the presence of the disulfide bond in the chimeric virus examined. Monoclonal and polyclonal antibodies directed against the V3 region of the HIV-1MN strain potently neutralized two chimeric viruses. Guinea pig antisera against two chimeric viruses were able to neutralize HIV-1MN and HIV-1ALA-1 in cell culture. The ability of chimeric viruses to elicit antibodies capable of neutralizing the source of the transplanted sequence could be favorable for vaccine development.

Animals↗
PubMed1999

New ways to evaluate chemotherapy competencies.

At a large teaching hospital with seven oncology units, nurse-educators implement a competency program to ensure that nurses with different backgrounds and patient populations master a unified set of skills and knowledge.

Antineoplastic Agents↗
PubMed1998

Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 A resolution.

The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the template-primer (including the primer grip, template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.

Amino Acid Sequence↗
PubMed1998

Structures of Tyr188Leu mutant and wild-type HIV-1 reverse transcriptase complexed with the non-nucleoside inhibitor HBY 097: inhibitor flexibility is a useful design feature for reducing drug resistance.

The second generation Hoechst-Bayer non-nucleoside inhibitor, HBY 097 (S-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydroqui noxalin-2(1H)-thione), is an extremely potent inhibitor of HIV-1 reverse transcriptase (RT) and of HIV-1 infection in cell culture. HBY 097 selects for unusual drug-resistance mutations in HIV-1 RT (e.g. Gly190Glu) when compared with other non-nucleoside RT inhibitors (NNRTIs), such as nevirapine, alpha-APA and TIBO. We have determined the structure of HBY 097 complexed with wild-type HIV-1 RT at 3.1 A resolution. The HIV-1 RT/HBY 097 structure reveals an overall inhibitor geometry and binding mode differing significantly from RT/NNRTI structures reported earlier, in that HBY 097 does not adopt the usual butterfly-like shape. We have determined the structure of the Tyr188Leu HIV-1 RT drug-resistant mutant in complex with HBY 097 at 3.3 A resolution. HBY 097 binds to the mutant RT in a manner similar to that seen in the wild-type RT/HBY 097 complex, although there are some repositioning and conformational alterations of the inhibitor. Conformational changes of the structural elements forming the inhibitor-binding pocket, including the orientation of some side-chains, are observed. Reduction in the size of the 188 side-chain and repositioning of the Phe227 side-chain increases the volume of the binding cavity in the Tyr188Leu HIV-1 RT/HBY 097 complex. Loss of important protein-inhibitor interactions may account for the reduced potency of HBY 097 against the Tyr188Leu HIV-1 RT mutant. The loss of binding energy may be partially offset by additional contacts resulting from conformational changes of the inhibitor and nearby amino acid residues. This would suggest that inhibitor flexibility can help to minimize drug resistance.

Antiviral Agents↗
PubMed1998

Genomic organization and cloning of the human homologue of murine Sipa-1.

Murine Sipa-1 (signal-induced proliferation associated protein) is a mitogen induced GTPase activating protein (GAP). While mapping candidate genes for multiple endocrine neoplasia type 1 (MEN1) at 11q13, we cloned the human homologue of Sipa-1. Herein, we report the complete cDNA sequence, expression, and genomic organization of SIPA-1. SIPA-1 consists of 16 exons with highly conserved exon-intron boundaries. The predicted SIPA-1 protein is highly homologous to the mouse protein, particularly in the region of the GAP-related domain at the amino terminus and the leucine zipper at the carboxy terminus. It is widely expressed, including in fetal tissues, but is most highly expressed in lymphoid organs. During the course of cloning SIPA-1, the MEN1 gene was identified, thus excluding human SIPA-1 as a candidate for this disease.

Amino Acid Sequence↗
PubMed1998

Effects of mutations in the polymerase domain on the polymerase, RNase H and strand transfer activities of human immunodeficiency virus type 1 reverse transcriptase.

Based on structural analyses and on the behavior of mutants, we suggest that the polymerase domain of HIV-1 reverse transcriptase (RT) plays a critical role in holding and appropriately positioning the template-primer both at the polymerase active site and at the RNase H active site. For RT to successfully copy the viral RNA genome, RNase H must cleave the RNA with absolute precision. We believe that a combination of the structure of the template-primer and its precise positioning are responsible for the specific cleavages RNase H makes. We have proposed that resistance of HIV-1 RT to nucleoside analogs involves a subtle repositioning of the template-primer. This hypothesis is based on both structural and biochemical analyses. Mutations that confer resistance to nucleoside analogs do not cluster at the polymerase active site; however, they are in positions where they could alter the interaction between RT and the template-primer. If, as we have hypothesized, the polymerase domain is primarily responsible for positioning the template-primer and RNase H cleavage depends on this positioning, it should be possible to use RNase H cleavage to monitor at least some of the major changes in the position of the template-primer. We have used three assays (polymerase, RNase H, and strand transfer) to investigate the effects of mutations in the polymerase domain, including mutations that confer resistance to nucleotide analogs, on HIV-1 RT. All three assays involve RNA sequences derived from the viral genome. The data show that alterations in the polymerase domain, in particular, mutations that are in positions that would be expected to alter the interaction of RT with the template-primer, can alter both the efficiency and specificity of RNase H cleavage. These results are discussed in light of the structure of HIV-1 RT.

Base Sequence↗
PubMed1998

Human rhinovirus type 14:human immunodeficiency virus type 1 (HIV-1) V3 loop chimeras from a combinatorial library induce potent neutralizing antibody responses against HIV-1.

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.

AIDS Vaccines↗