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E J Arts

Publications and source records attributed to E J Arts.

At least 19 recordsLinked to original sources
PubMed2006

Virus fitness: concept, quantification, and application to HIV population dynamics.

Viral fitness has been broadly studied during the past three decades, mainly to test evolutionary models and population theories difficult to analyze and interpret with more complex organisms. More recent studies, however, are focused in the role of fitness on viral transmission, pathogenesis, and drug resistance. Here, we used human immunodeficiency virus (HIV) as one of the most relevant models to evaluate the importance of viral quasispecies and fitness in HIV evolution, population dynamics, disease progression, and potential clinical implications.

Anti-HIV Agents↗
PubMed2001

Functional characterization of chimeric reverse transcriptases with polypeptide subunits of highly divergent HIV-1 group M and O strains.

Human immunodeficiency virus (HIV)-1 strains have been divided into three groups: main (M), outlier (O), and non-M non-O (N). Biochemical analyses of HIV-1 reverse transcriptase (RT) have been performed predominantly with enzymes derived from HIV-1 group M:subtype B laboratory strains. This study was designed to optimize the expression and to characterize the enzymatic properties of HIV-1 group O RTs as well as chimeric RTs composed of group M and O p66 and p51 subunits. The DNA-dependent DNA polymerase activity on a short heteropolymeric template-primer was similar with all enzymes, i.e. the HIV-1 group O and M and chimeric RTs. Our data revealed that the 51-kDa subunit in the chimeric heterodimer p66(M:B)/p51(O) confers increased heterodimer stability and partial resistance to non-nucleoside RT inhibitors. Chimeric RTs (p66(M:B)/p51(O) and p66(O)/p51(M:B)) were unable to initiate reverse transcription from tRNA(3)(Lys) using HIV-1 group O or group M:subtype B RNA templates. In contrast, HIV-1 group O and M RTs supported (-)-strand DNA synthesis from tRNA(3)(Lys) hybridized to any of their corresponding HIV-1 RNA templates. HIV-2 RT could not initiate reverse transcription on tRNA(3)(Lys)-primed HIV-1 genomic RNA. These findings suggest that the initiation event is conserved between HIV-1 groups, but not HIV types.

Base Sequence↗
PubMed2001

Mechanisms involved in stimulation of human immunodeficiency virus type 1 replication by aminooxypentane RANTES.

Aminooxypentane (AOP)-RANTES is a potent inhibitor of nonsyncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) isolates. Although classical chemotactic responses are not induced in primary leukocytes by AOP-RANTES, recent studies suggest that a remnant of cell signaling occurs upon binding of receptor to this compound. We have detected a breakthrough of NSI/R5 replication from the inhibitory effects of high AOP-RANTES concentrations (<100 nM). A stimulation of different primary syncytium-inducing (SI), CXCR4-tropic (X4) HIV-1 isolates was also observed in the presence of AOP-RANTES. This stimulation was also observed after 110 h in PCR and RT-PCR for minus-strand strong-stop DNA and unspliced and multiply spliced RNA, respectively. However, there was significant variability between different SI/X4 or NSI/R5 HIV-1 isolates with regard to this AOP-RANTES-mediated stimulation or breakthrough, respectively. To further define the mechanism(s) responsible for this AOP-RANTES effect, we performed detailed retroviral replication studies with an NSI/R5 (B-92BR021) and SI/X4 (D-92UG021) HIV-1 isolate in the presence of the drug. Treatment of peripheral blood mononuclear cells with 125 nM AOP-RANTES and virus did not alter coreceptor expression, HIV-1 entry, reverse transcription, or mRNA transcription from the long terminal repeat, but it did result in increased HIV-1 integration. This AOP-RANTES-mediated increase in HIV-1 integration was diminished by treatment with pertussis toxin. Phosphorylation of the mitogen-activated protein kinase (MAPK) isoforms, extracellular signal-regulated kinase 1 (ERK1) and ERK2, was increased in a CD4(+) CCR5(+) U87 cell line treated with AOP-RANTES or with an NSI/R5 HIV-1 isolate. These findings suggest that AOP-RANTES may induce a MAPK/ERK signal transduction pathway upon binding to a G-protein-coupled receptor. MAPK/ERK1 and -2 appear to phosphorylate the HIV-1 preintegration complex, a step necessary for nuclear translocation and successful integration.

Anti-HIV Agents↗
PubMed2000

Greater diversity of HIV-1 quasispecies in HIV-infected individuals with active tuberculosis.

OBJECTIVE: A continual increase in intrapatient HIV-1 heterogeneity is thought to contribute to evasion of host immune response and eventual progression to AIDS. Tuberculosis (TB) is diagnosed both early and late during the course of HIV-1 disease and may increase diversity of HIV-1 quasispecies by activating the HIV-1 immune response and increasing HIV-1 replication. We examined whether HIV-1 heterogeneity is altered in HIV-1-infected individuals with TB. METHODS: Blood samples were obtained from 7 HIV-1-infected patients with active TB (HIV/TB patients) and 9 HIV-1-infected patients (HIV patients) in Kampala, Uganda (CD4 counts of 0-650 cells/microl and HIV loads of 700-750,000 RNA copies/ml). The C2-C3 region of the HIV-1 envelope gene (env) was amplified by nested polymerase chain reaction (PCR) from lysed peripheral blood mononuclear cells (PBMCs) of each patient, and then subject to sequencing, clonal-quasispecies analysis and heteroduplex tracking analysis (HTA). RESULTS: HTA of env DNA fragments showed increased heterogeneity in the HIV/TB individuals compared with the HIV group. Further sequence and HTA analysis on ten individual env clones for each patient showed significantly greater HIV mutation frequencies in HIV/TB patients than in HIV patients. CONCLUSION: An increase in HIV-1 heterogeneity may be associated with a TB-mediated increase in HIV-1 replication. However, a diverse HIV-1 quasispecies population in HIV/TB patients as opposed to tight quasispecies clusters in HIV patients suggests a possible dissemination of lung-derived HIV-1 isolates from the TB-affected organ.

Adult↗
PubMed2000

Quantitation of human immunodeficiency virus type 1 group O load in plasma by measuring reverse transcriptase activity.

We have evaluated the use of an ultrasensitive reverse transcriptase (RT) activity assay to monitor plasma viremia in two human immunodeficiency virus type 1 (HIV-1) group O-infected patients treated with stavudine, lamivudine, and indinavir. After a initial decline in RT levels observed at 4 weeks of therapy, RT-based plasma viremia returned to baseline values at 28 or 44 weeks of treatment. The rebound in levels of RT activity was associated with the detection of phenotypic resistance to lamivudine and with the Met184Val mutation. Analysis of RT activity in plasma provides a sequence-independent means of monitoring virus loads in HIV-1 group O-infected patients.

Adult↗
PubMed2000

Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs.

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other beta-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC(50)) ranged from 0.04 nM with HIV-1(A-92RW009) to 1.3 nM with HIV-1(B-BaL). Experiments performed on the same isolate (HIV-1(B-BaL)) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC(50) values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC(50) values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.

Amino Acid Sequence↗
PubMed2000

A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression.

This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4(+) T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

Biological Assay↗
PubMed1998

Mutating a region of HIV-1 reverse transcriptase implicated in tRNA(Lys-3) binding and the consequences for (-)-strand DNA synthesis.

Recently, tRNALys-3 was cross-linked via its anticodon loop to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) between residues 230 and 357 (Mishima, Y., and Steitz, J. A. (1995) EMBO J. 14, 2679-2687). Scanning the surface of this region identified three basic amino acids Lys249, Arg307, and Lys311 flanking a small crevice on the p66 thumb subdomain outside the primer-template binding cleft. To assess an interaction of this region with the tRNA anticodon loop, these p66 residues were altered to Glu or Gln. p66 subunits containing K249Q, K311Q, K311E, and a dual R307E/K311E mutation formed a stable dimer with wild type p51. All mutants showed reduced affinity for tRNALys-3 and supported significantly less (-)-strand DNA synthesis from this primer than the parental heterodimer. In contrast, these variants efficiently synthesized HIV-1 (-)-strand strong-stop DNA from oligonucleotide primers and had minimal effect on RNase H activity, retaining endonucleolytic and directed cleavage of an RNA/DNA hybrid. Structural features of binary RT.tRNALys-3 complexes were examined by in situ footprinting, via susceptibility to 1, 10-phenanthroline-copper-mediated cleavage. Unlike wild type RT, mutants p66(K311Q)/p51 and p66(K311E)/p51 failed to protect the tRNA anticodon domain from chemical cleavage, indicating a significant structural alteration in the binary RT.tRNA complex. These results suggest a crevice in the p66 thumb subdomain of HIV-1 RT supports an interaction with the tRNALys-3 anticodon loop critical for efficient (-)-strand DNA synthesis.

Anticodon↗
PubMed1998

Interaction of retroviral reverse transcriptase with template-primer duplexes during replication.

Conversion of the single-stranded RNA of an invading retrovirus into double-stranded proviral DNA is catalyzed in a multi-step process by a single virus-coded enzyme, reverse transcriptase (RT). Achieving this requires a combination of DNA polymerase abd ribonuclease H (RNase H) activities, which are located at the amino and carboxy terminus of the enzyme, respectively. Moreover, proviral DNA synthesis requires that three structurally-distinct nucleic acid duplexes are accommodated by this enzyme, namely (a) A-form RNA (initiation of minus strand synthesis), non-A, non-B RNA/DNA hybrid (minus strand synthesis and initiation of plus strand synthesis) and B-form duplex DNA (plus strand synthesis). This review summarizes our current understanding of the manner in which retroviral RT interacts with this diverse array of nucleic acid duplexes, exploiting in many cases mutants unable to catalyze a specific event. These studies illustrate that seemingly 'simple' events such as tRNA-primed initiation of minus strand synthesis are considerably more complex, involving intermolecular tRNA-viral RNA interactions outside the primer binding site. Moreover, RNase H activity, generally thought to catalyze non-specific degradation of the RNA-DNA replicative intermediate, is required for highly specialized events including DNA strand transfer and polypurine selection. Finally, a unique structure near the center of HIV proviral DNA, the central termination sequence, serves to halt the replication machinery in a manner analogous to termination of transcription. As these highly specialized events are better understood at the molecular level, they may open new avenues of therapeutic intervention in the continuing effort to stem the progression of HIV infection and AIDS.

Base Sequence↗
PubMed1998

Mechanisms of clinical resistance by HIV-I variants to zidovudine and the paradox of reverse transcriptase sensitivity.

Even with the development of novel nucleoside analog inhibitors, zidovudine (AZT or 3'-azido-3'-deoxythymidine) remains a potent and frequently prescribed antiretroviral therapy for HIV-positive individuals. Failure of AZT in monotherapy due to the emergence of drug-resistant virus has not excluded it from use in most combination therapies with other nucleoside analogs, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Thus, an understanding of the mechanism of AZT resistance could be the key in predicting the failure of many treatment strategies. In this review, the occurrence, characterization and ramification of AZT resistance in HIV-positive individuals will be discussed in the context of genotypic and phenotypic analyses of AZT-resistant viruses and reverse transcriptases. The mechanisms of resistance to AZT may be distinct from the mechanisms of resistance to other nucleoside analogs.

Journal Article↗
PubMed1998

Analysis of pol gene heterogeneity, viral quasispecies, and drug resistance in individuals infected with group O strains of human immunodeficiency virus type 1.

Nucleotide sequences of the reverse transcriptase (RT) coding region have been compared in four new human immunodeficiency virus type 1 (HIV-1) group O isolates. Phylogenetic analysis of this pol region highlights a cluster of these four HIV-1 group O sequences with seven other group O isolates (5% intracluster nucleotide sequence diversity) similar to clusters classified as subtypes in HIV-1 group M (an average of 4.9% intrasubtype sequence diversity). Based on these analyses, this group O cluster has been designated subtype A-O. A longitudinal study of a heterosexual couple infected with group O (ESP1 and ESP2) allowed a detailed analysis of RT sequences (amino acids 28 to 219). Directed evolution and a slightly higher mutation frequency was observed in the RT sequences of patient ESP2, treated with antiretroviral drugs, than that from the untreated patient ESP1. Antiretroviral treatment also selected for specific substitutions, M184V and T215Y in the RT coding region, conferring resistance to 3'-dideoxy-3'-thiacytidine and zidovudine, respectively. A Gly98 to Glu RT substitution identified in the treated patient suggests a possible reversion of a nonnucleoside RT inhibitor-resistant phenotype. Using RT clones from this longitudinal study, both heteroduplex tracking assay and cloning-sequencing techniques were employed for an extensive genetic analysis of pol gene quasispecies. Amino acid substitutions (i.e., Phe-77 to Leu, Lys-101 to Glu, and Val-106 to Iso) associated with antiretroviral resistance were identified in RT clones from HIV-1 group O-infected patients not subjected to drug therapy or treated with unrelated drugs. Finally, phylogenetic relationships between RT clones of the treated ESP2 patient and those of the untreated ESP1 patient show how drug pressure can direct evolution of viral pol gene quasispecies independently of direct drug-resistant substitutions.

Adolescent↗
PubMed1998

3'-Azido-3'-deoxythymidine (AZT) mediates cross-resistance to nucleoside analogs in the case of AZT-resistant human immunodeficiency virus type 1 variants.

Difficulties in deciphering the mechanisms of 3'-azido-3'-deoxythymidine (AZT)-resistance by human immunodeficiency virus type 1 (HIV-1) variants are due in part to an inability to reconstitute resistance in vitro using AZT-resistant reverse transcriptases. We decided to characterize mechanisms of AZT resistance in tissue culture infections by studying the ability of drug-resistant viruses to synthesize viral DNA in the presence or absence of drug. Through use of PCR amplifications, we discovered an AZT-mediated stimulation of reverse transcription by AZT-resistant viruses carrying the M41L and T215Y mutations that can apparently override the inhibitory effects of AZT-5'-triphosphate. In addition, the presence of AZT also causes viruses containing the M41L and T215Y substitutions to have diminished sensitivity to other nucleoside analogs (i.e., ddC, ddI, and d4T). This AZT-mediated cross-resistance may help to explain the virological failure of treatment regimens that included ddI plus AZT or ddC plus AZT in situations in which the T215Y and/or M41L mutations were present (F. Brun-Vézinet, C. Boucher, C. Loveday, D. Descamps, V. Fauveau, J. Izopet, D. Jeffries, S. Kaye, C. Krzyanowski, A. Nunn, R. Schuurman, J. M. Seigneurin, C. Tamalet, R. Tedder, J. Weber, and G. J. Weverling, Lancet 350:983-990, 1997). Our results suggest that the use of AZT may be contraindicated in those patients for whom resistance to this compound (M41L and/or T215Y) has been demonstrated.

Anti-HIV Agents↗
PubMed1996

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Animals↗
PubMed1996

Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication.

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.

Base Sequence↗
PubMed1996

Involvement of C-terminal structural elements of equine infectious anemia virus reverse transcriptase in DNA polymerase and ribonuclease H activities.

In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcriptase (EIAV RT), recombinant p66 polypeptides containing a modified ribonuclease H (RNase H) domain were purified and evaluated. Defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of DNA polymerase and RNase H activities, while DNase I footprinting revealed features of replication complexes containing the truncated enzymes. Removal of alpha-helix E' and the conserved beta 5'-alphaE' "His-loop" in p66delta20 RT uncouples the RNase H activities, alters affinity for template-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, DNase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-terminal residues, which includes alpha-helix D', beta-strand 5' and alpha-Helix E', yields an enzyme with distributive DNA polymerase properties closely resembling the purified p51 subunit.

Amino Acid Sequence↗