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Biomedical subjects

A M Lever

Publications and source records attributed to A M Lever.

At least 19 recordsLinked to original sources
PubMed2001

Oligonucleotide mapping of the core genomic RNA dimer linkage in human T-cell leukaemia virus type-1.

We have previously mapped the sequences required for dimerisation of the 5' leader of the human T-cell leukaemia virus type-1 (HTLV-1) genome. The smallest sequence necessary and sufficient for dimer formation, in vitro, was ascertained to be a 37 nucleotide (nt) region downstream of the splice donor and just upstream of the primer binding site. Deletion of a 32 base-pair sequence encompassing this region within the provirus was associated with a minor decrease in infectivity of the virus in an in vitro system. To further map and help elucidate the nature of the dimer linkage, we used RNA and DNA oligonucleotide competition assays to define the nucleotides involved. These experiments revealed that a 14 nt sequence containing a potential stem loop structure, formed from a palindromic sequence, is important for dimer formation. This was confirmed by the ability of this RNA sequence to form heterodimers with larger RNA transcripts from the same region, while sequences lacking this motif could not. RNA transcripts containing the reverse sequence, the same nucleotides in a random arrangement, and complementary DNA oligos, all failed to form heterodimers with the 14 nt sequence. The primary dimer initiation site of HTLV-1 has thus been located to a 14 nt palindrome containing sequence, and dimerisation is shown to be dependent on specific sense-sense RNA interactions.

Computer Simulation↗
PubMed2001

Sequences in the 5' leader of Mason-Pfizer monkey virus which affect viral particle production and genomic RNA packaging: development of MPMV packaging cell lines.

We used a series of deletion mutations in the 5' untranslated region of the prototype D type retrovirus, Mason-Pfizer Monkey Virus (MPMV), to analyse RNA encapsidation. A region was identified upstream of the major splice donor which reduced particle production but had a proportionally greater effect on RNA packaging. A small deletion downstream of the splice donor had little effect on RNA production and caused no significant packaging defect. A large deletion encompassing the end of the primer binding site down to the splice donor had a dramatic effect, disrupting viral protein synthesis. Stable cell lines were produced containing packaging-defective virus. These first-generation packaging cell lines were used to package and transfer an MPMV-based vector.

5' Untranslated Regions↗
PubMed2001

Chromosomal positioning of human T-lymphotropic type 1 proviruses by fluorescence in situ hybridisation.

Fluorescence in situ hybridisation (FISH) was employed to identify the chromosomal integration site of the human T-cell lymphotropic virus, type 1 (HTLV-1) present in T-cell clones derived from HTLV-1-infected individuals and a virally transformed cell line, C8166-45. Proviral sequences were detected in C8166-45 but not uninfected Jurkat cells. Integration sites were reliably detected in T-cell clones determined previously to be infected with HTLV-1. The results indicated that the transformed cell line and some of the T-cell clones possessed more than one proviral integration site. This hybridisation system is useful for determining the number of integration events and for localising proviruses to specific chromosomal regions.

Cell Line, Transformed↗
PubMed2001

Comparative antimicrobial efficacy of multi-purpose hydrogel lens care solutions.

Ten single-bottle multipurpose hydrogel lens care solutions commercially available in the European market were evaluated for antimicrobial efficacy using the International Organization for Standardization's (ISO) Stand-Alone Procedure. The results of this study indicate that ReNu Multi Plus ReNu Multi Purpose, Complete and DUA meet the ISO Stand-Alone primary acceptance criteria for stand-alone disinfectants against all challenge organisms: Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa, Candida albicans, and Fusarium solani. Solo-Care Soft, All-In-One Light, Optiplus, Opti Free Express, UniCare and Combi Comfort do not meet the ISO Stand-Alone primary acceptance criteria for one or more test organisms within their respective labelled minimum disinfection times. In addition, although not a requirement of the guidance document, only ReNu MultiPlus and ReNu Multi-Purpose exceeded the minimum ISO Stand Alone primary acceptance criteria within 25% (1 h) of their labelled minimum disinfection time for all test organisms. This evaluation provides a direct comparison of antimicrobial activity for commercially available multipurpose lens care solutions at their labelled minimum disinfection times. The results of this study should be considered when selecting appropriate lens care systems for patients.

Journal Article↗
PubMed2001

Investigation of RNA transcripts containing HIV-1 packaging signal sequences as HIV-1 antivirals: generation of cell lines resistant to HIV-1.

Based on the success of RNA decoy approaches using RRE and TAR sequences to inhibit HIV-1 replication, we studied the ability of HIV-1 packaging signal sequences to interfere with viral RNA encapsidation and formation of infectious particles. We made a variety of plasmid constructs in which the sequence context or number of repeats of the viral packaging signal was varied, and investigated the ability of these transcripts to inhibit replication of HIV-1 in stably transfected Jurkat T lymphocytes. We found that certain lines showed strong inhibition of HIV-1 replication, an effect that persisted at high input amounts of virus and significantly delayed viral replication for up to 4 weeks. An investigation of the mechanism of inhibition revealed that in these cell lines the packaging efficiency of the genomic HIV-1 transcript was unaffected. Further studies identified an antiviral effect on both HIV-1 and HIV-2 that did not correlate with decoy expression, and was substantially independent of CD4 expression or cellular proliferative capacity. Study of these resistant cell lines may lead to new insights into mechanisms of inhibition of HIV-1 replication.

CD4 Antigens↗
PubMed2001

The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity.

Deletion of a region of the human immunodeficiency virus type 2 (HIV-2) 5' leader RNA reduces genomic RNA encapsidation to about 5% that of wild-type virus with no defect in viral protein production but severely limits virus spread in Jurkat T cells, indicating that this region contains a major cis-acting encapsidation signal, or psi (Psi). Being upstream of the major splice donor, it is present on all viral transcripts. We have shown that HIV-2 selects its genomic RNA for encapsidation cotranslationally, rendering wild-type HIV-2 unable to encapsidate vector RNAs in trans. Virus with Psi deleted, however, encapsidates an HIV-2 vector, demonstrating competition for Gag protein. HIV-2 overcomes the lack of packaging signal location specificity by two novel mechanisms, cotranslational packaging and competition for limiting Gag polyprotein.

Animals↗
PubMed2000

HIV RNA packaging and lentivirus-based vectors.

Since the mid-1990s, the number of publications on lentivirus-based vectors has expanded dramatically as people have realized the opportunity that they represent. High-titer helper-virus free transfer of genes to nondividing cells is a reality and it can only be a short time before clinical trials are initiated. The most efficient vector to date appears to be HIV-1 and it is no coincidence that this is the virus in which there is the greatest theoretical understanding of the encapsidation process and viral assembly. Basic studies in the other viruses are at an earlier stage and this is reflected to some extent in their relative inefficiency. Emphasis is placed in some publications on non-HIV-based vector systems having the additional safety feature of a viral vector not based on a human pathogen. As yet, this is largely a cosmetic advantage in that no system would be used which was capable of regenerating a full-length wild-type HIV and the vectors all have single round replication kinetics. More important will be elucidation of the mechanism of packaging in the different lentiviruses. Cis and trans packaging preferences may influence efficiency. Accurate delineation of packaging signals will be important. Most influential, however, will be a deeper understanding of all the viral and cellular factors involved in the packaging pathway.

Base Sequence↗
PubMed2000

Antisense RNA sequences targeting the 5' leader packaging signal region of human immunodeficiency virus type-1 inhibits viral replication at post-transcriptional stages of the life cycle.

Antisense RNA has proven a potent inhibitor of gene expression and has the potential to inhibit retroviral replication at a number of stages in the virus life cycle by targeting both viral and cellular RNA sequences. Antisense RNA complementary to three target regions in the 5' leader/LTR of human immunodeficiency virus type-1 (HIV-1), the TAR region, the primer binding site and the splice donor (SD)-packaging signal (psi) region were stably expressed from the CMV IE promoter in Jurkat cells, and expression confirmed by RT-PCR. When challenged with HIV-1, cell lines expressing antisense RNA targeting the SD/psi region showed significant inhibition of replication (at up to 10(6) TCID 50/ml). These sequences were also expressed in lymphocytes after transduction using recombinant retroviruses and one sequence complementary to the SD/psi region inhibited replication of HIV-1. A co-transfection assay using COS-1 cells was also developed both to confirm the antiviral potential of these sequences, and to determine the predominant site of action of these molecules. Antisense RNAs targeting the psi region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore, analyses of relative levels of cellular and virion RNA from these assays suggest each of these antisense molecules exerts its effect at an early stage in the transcription-translation pathway, while the longer of the sequences also inhibited packaging of virion RNA. These results suggest that the packaging signal (psi) of HIV-1 represents an attractive target for antisense RNA-based gene therapy, although the main mode of action of such molecules may well be through antisense effects at an earlier stage of replication than packaging.

3T3 Cells↗
PubMed2000

A 37 base sequence in the leader region of human T-cell leukaemia virus type I is a high affinity dimerization site but is not essential for virus replication.

Mutagenesis has demonstrated a region in the human T-cell leukaemia virus type I (HTLV-I) 5' leader RNA which, when deleted, abolishes stable RNA dimer formation in vitro. We have further mapped, using both in vitro transcribed and synthesized RNA, this site to a 37 base region, which dimerizes with high affinity. When deleted from an HTLV-I Gag-Pol-expressing plasmid which was co-transfected with an envelope protein expressor to produce virions capable of single round infection, the dimer linkage deletion did not affect viral protein production. In addition, virus infectivity was only slightly reduced, to approximately 75-80% of the wild-type.

5' Untranslated Regions↗
PubMed2000

Virus inactivation in a proportion of human T-cell leukaemia virus type I-infected T-cell clones arises through naturally occurring mutations.

Human T-cell leukaemia virus type I (HTLV-I) is the aetiological agent of adult T-cell leukaemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). The trans-activating protein (Tax) of HTLV-I is strongly implicated in cellular proliferation. We examined the tax gene and 5' long terminal repeat (LTR) sequences in eight naturally infected T-cell clones derived from TSP/HAM-affected individuals who were either productively (proliferate spontaneously) or silently (do not proliferate spontaneously) infected. In two silently infected clones point mutations within the proviruses resulted in truncation of the Tax protein. One clone harboured both a deleterious tax gene mutation and a point mutation in an enhancer element of the 5' LTR. Sequence changes, immunological escape mutation, integration site context and host cell phenotype may all contribute to the high proportion of latently or silently infected T-cells found in vivo in virus carriers.

5' Untranslated Regions↗
PubMed2000

Lentiviral vectors: progress and potential.

Lentiviral vectors are continuing to generate great interest as gene vectors for possible gene therapy in vivo. Over the past year, considerable progress has been made in demonstrating gene delivery to a wide variety of tissues, taking advantage of their unique capability of integrating the desired gene into the chromosomes of the target cell. The basic science of lentivirus vector production has expanded and their relative ease of utility is reflected by the broadening range of laboratories reporting successful gene transfer using them. Important new findings on RNA processing within the cell have come from studies of vectors which have direct relevance to the molecular biology of HIV itself. Although none have been used in clinical trials in vivo, it can only be a matter of time before this occurs. This article reviews publications over the last 12 months, highlighting the major achievements in this field.

Animals↗
PubMed1999

Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation.

Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.

Amino Acid Sequence↗
PubMed1999

Molecular investigations implicate human endogenous retroviruses as mediators of anti-retroviral antibodies in autoimmune rheumatic disease.

Polymerase chain reaction using specific primers, failed to detect HTLV-I amplicons in patients with rheumatic diseases previously shown to possess antibodies to retroviral products. However, by employing broad spectrum oligonucleotide primers, 135 bp amplicons were generated from peripheral blood mononuclear cells and synovial fluid cells. Subsequent cloning and DNA sequencing revealed homology to a number of exogenous and human endogenous retroviruses (HERVs). Furthermore, in combining the presence of type B and C related endogenous retroviruses, a significant association (p=0.014) was apparent for chronic autoimmune rheumatic diseases as compared to controls. Reverse transcription polymerase chain reaction of RNA derived from patients, healthy controls and cell lines (U937, BJAB, human endothelial lung fibroblasts) demonstrated ubiquitous expression of HERV-K10 and RTVL-H2. Furthermore messenger RNA expression of HERV-K10 was enhanced in fibroblasts infected with human cytomegalovirus. It is plausible that subsequent production of HERV peptides could explain the presence of anti-retroviral antibodies in cohorts of patients with autoimmune rheumatic diseases.

Antibodies, Viral↗