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T Middleton

Publications and source records attributed to T Middleton.

12 recordsLinked to original sources
PubMed2000

Evidence for late stage compartmentalization of HIV-1 resistance mutations between lymph node and peripheral blood mononuclear cells.

OBJECTIVE: To determine the overall distribution of drug-resistance mutations to nucleoside reverse transcriptase inhibitors of HIV strains recovered from the lymph nodes (LN) and peripheral blood mononuclear cell (PBMC) compartments of four HIV-infected patients receiving zidovudine and didanosine and to compare them with antiretroviral-naive patients. DESIGN: Molecular comparison of major and minor HIV-1 env and pol region variants residing in LN and PBMC compartments. MATERIALS AND METHODS: Proviral DNA sequences were amplified by PCR from both PBMC and LN compartments, cloned into PGEM-T II Easy vector and sequenced. The clones were subjected to molecular and phylogenetic analysis. RESULTS: Comparison of PBMC and LN-derived HIV-1 variants in the env V3 region showed that nucleotide and amino acid variability was a characteristic feature of LN-derived variants. In contrast, a majority of resistance mutations to reverse transcriptase inhibitors were localized in the PBMC compartment rather than in LN, which is thought to be a reservoir of HIV. CONCLUSIONS: Distinct compartmentalization or independent evolution of pol and env gene variants between LN and PBMC could be due to the differential selection pressure imposed by the combination drug regimen, hence the bimodal distribution of resistance variants between LN and PBMC compartments.

Anti-HIV Agents↗
PubMed1998

Stability of human immunodeficiency virus RNA in blood specimens as measured by a commercial PCR-based assay.

We investigated the effects of conditions often encountered during handling, transit, and storage of blood specimens on the quantity of detectable human immunodeficiency virus (HIV) RNA in plasma. HIV RNA copy numbers were measured with a commercially available assay (the Amplicor HIV-1 Monitor test kit). Variables examined were the time to processing of blood and plasma, the holding temperature of blood and plasma prior to processing, the effect of freezing and thawing of plasma, and the use of different anticoagulants. The relationship between the HIV RNA copy number and the HIV isolation rate by peripheral blood mononuclear cell (PBMC) coculture was also examined. We found that RNA copy numbers were maintained to within 0.5 log10 (approximately threefold) in blood and plasma samples held at room temperature or 4 degrees C for up to 3 days and remained stable despite (limited) freezing and thawing of the plasma. HIV RNA copy numbers were also maintained after long-term storage of plasma at -70 degrees C. The ability to isolate HIV from PBMCs was directly proportional to the HIV RNA copy number.

Adolescent↗
PubMed1997

NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein.

The E2 protein is required for the replication of human papillomaviruses (HPVs), which are responsible for anogenital warts and cervical carcinomas. Using an NMR-based screen, we tested compounds for binding to the DNA-binding domain of the HPV-E2 protein. Three classes of compounds were identified which bound to two distinct sites on the protein. Biphenyl and biphenyl ether compounds containing a carboxylic acid bind to a site near the DNA recognition helix and inhibit the binding of E2 to DNA. Benzophenone-containing compounds which lack a carboxylic acid group bind to the beta-barrel formed by the dimer interface and exhibit negligible effects on the binding of E2 to DNA. Structure-activity relationships from the biphenyl and biphenyl ether compounds were combined to produce a compound [5-(3'-(3",5"-dichlorophenoxy)-phenyl)-2,4-pentadienoic acid] with an IC50 value of approximately 10 microM. This compound represents a useful lead for the development of antiviral agents that interfere with HPV replication and further illustrates the usefulness of the SAR by NMR method in the drug discovery process.

Antiviral Agents↗
PubMed1995

Multiple regions within EBNA1 can link DNAs.

Epstein-Barr virus nuclear antigen 1 (EBNA1) can bind specifically to two clusters of sites within the Epstein-Barr virus plasmid origin of DNA replication (oriP). EBNA1 activates DNA replication mediated by oriP and can also activate transcription and retain DNA in cells when bound site specifically. EBNA1 bound to oriP physically links the two clusters of EBNA1-binding sites, resulting in loop formation by the intervening DNA. To elucidate the contribution of DNA linking by EBNA1 to its biological activities, we identified regions within it that can independently link DNAs to which they are bound. An electrophoretic mobility shift assay was used to detect this activity. Proteins which link DNA aggregate that DNA into large lattices. Proteins which cannot link DNA but still bind to DNA retard the mobility of that DNA but do not cause it to form lattices. Amino-terminal truncations were used to map the amino-terminal limit of a minimal DNA-linking domain approximately to amino acid 372 of EBNA1. To map the carboxy-terminal limit of this minimal domain, fusion proteins containing the DNA-binding domain of GAL4 and fragments of EBNA1 were generated and studied. This approach identified the carboxy-terminal limit of this minimal domain to be approximately amino acid 391 and verified its amino-terminal limit. Internal deletions within a truncated EBNA1 derivative verified the importance of this region. Two additional fragments of EBNA1, each of which independently conferred DNA-linking activity on the domain of GAL4 which binds DNA, were identified within amino acids 54 to 89 and amino acids 331 to 361. Therefore, EBNA1 contains at least three regions that can act independently to link DNAs and that may act in concert within intact EBNA1.

Amino Acid Sequence↗
PubMed1994

Retention of plasmid DNA in mammalian cells is enhanced by binding of the Epstein-Barr virus replication protein EBNA1.

The capacity to bind the Epstein-Barr viral protein EBNA1 increases the retention of the plasmid in dividing cells. This retention requires binding of multiple EBNA1 molecules for function, although significant retention activity is seen with fewer EBNA1 binding sites than are required to activate replication or transcription. The regions of EBNA1 that are required for increased plasmid retention overlap with those required for activation of transcription and replication. The similarities in traits of EBNA1 that are required for support of DNA replication and retention of plasmid DNA indicate that both may be mediated by interactions with an overlapping set of cellular proteins.

Antigens, Viral↗
PubMed1992

EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication.

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.

Adenosine Triphosphatases↗
PubMed1992

A chimera of EBNA1 and the estrogen receptor activates transcription but not replication.

DNA replication from the plasmid origin of replication of Epstein-Barr virus requires one viral protein, EBNA1. This protein also acts as a transcriptional activator. Mutational analyses of EBNA1 have led to the conclusion that it supports transcription and DNA replication similarly. Such analyses have not probed the DNA-binding domain of EBNA1. To test whether domains of EBNA1 specifically required for either transcription or replication lie within its DNA-binding domain, we constructed a functional transcriptional activator by placing the EBNA1 DNA-binding domain in the context of the activation domains of the estrogen receptor. This hybrid protein did not support DNA replication, which indicates that the DNA-binding domain does not contain a replication-specific domain that can function along with heterologous transcriptional activating domains.

Amino Acid Sequence↗
PubMed1991

DNA looping between the origin of replication of Epstein-Barr virus and its enhancer site: stabilization of an origin complex with Epstein-Barr nuclear antigen 1.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support replication of Epstein-Barr virus during the latent phase of its life cycle. The DNA segment required for latent replication, oriP, contains two essential binding regions for EBNA-1, termed FR and DS, that are separated by 1 kilobase pair. The FR site appears to function as a replicational enhancer providing for the start of replication at the DS site. We have used electron microscopy to visualize the interaction of EBNA-1 with its binding sites and to study the mechanism for communication between the FR and DS sites. We have found that DNA-bound EBNA-1 forms a DNA loop between the FR and DS sites. From these results, we suggest that EBNA-1 bound to the replicational enhancer acts by a DNA-looping mechanism to facilitate the initiation of DNA replication. Occupancy of the DS site alone is highly sensitive to competition with nonspecific DNA. In contrast, occupancy of the DS site by looping from FR is largely resistant to the competitor DNA. These experiments support the concept that enhancers act in cis from nearby sites to provide a high local concentration of regulatory proteins at their target sites and to stabilize regulatory interactions.

Animals↗
PubMed1991

Immortalizing genes of Epstein-Barr virus.

EBV immortalizes human B lymphocytes efficiently. Ten of its approximately 100 genes are expressed in these proliferating lymphoblasts and are candidates for mediating the changes central to the immortalization of the cell. Enough has been learned now about three of these viral genes to indicate that they are likely to be required for immortalization. As more is learned, additional genes of EBV will probably be found to support the process of immortalization of the host cell. EBNA-2 has been shown genetically to be required for EBV to immortalize an infected B lymphocyte. The biochemical activities of EBNA-2 that constitute this requirement have not been identified. Many experiments indicate that EBNA-2 affects the accumulation of specific viral and cellular RNAs. These effects, however, can be detected only in certain EBV-negative B-lymphoblastoid cells. It is, therefore, not clear that the known effects of EBNA-2 adequately explain its ubiquitous requirement in the immortalization of primary human B lymphocytes. LMP is likely to be required for immortalization because it can affect the growth properties of established human lymphoid and epithelial cells and can transform at least two established rodent cells to proliferate in an anchorage-independent manner. The structure of this viral protein, its position in the plasma membrane, many of its biochemical properties, as well as studies of its mutant derivatives are consistent with its acting as a growth factor receptor or affecting the activity of such a receptor. However, no biochemical activity has been assigned directly to LMP, and both its mechanism of action and its possible contribution to immortalization by EBV remain enigmatic. EBNA-1 presumably is required for EBV to immortalize a B lymphocyte because it is essential for the initiation of plasmid DNA replication by EBV. Circumstantial observations indicate also that EBNA-1 is probably necessary for sustaining viral DNA replication in the proliferating cell population. EBNA-1 may well affect the regulation of transcription of viral genes that themselves are required for immortalization. These roles of EBNA-1 are performed in part by its site-specific binding to the elements of oriP required in cis for the replication of EBV plasmid DNAs. It is probable that EBNA-1 also binds both to a set of cellular proteins that function in transcription and to a nonidentical set of cellular proteins that function in replication. EBV effects a fascinating phenotypic change in B lymphocytes it infects. It does so by using several viral genes that alter the physiology of the cell by different means.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.

Antibodies, Viral↗
PubMed1985

Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography.

T4 RNA ligase has been used to construct a series of defined oligoribonucleotides. Hexamer or pentamer blocks were synthesized first by multiple additions of mononucleotide diphosphates to trimers with T4 RNA ligase and removal of the terminal phosphate with alkaline phosphatase; inhibitors of the ligase were removed by passing the sample over a 1-ml reverse-phase octadecasilyl column. The two nucleotide blocks were then ligated to give undecamers. Yields for the individual ligations ranged from 85 to 100% for acceptors lacking uridines and at least 70% for those containing uridines. The overall yield of the undecamer relative to the starting trimers was about 10%. Each round of ligation averaged about 8 h; the time required to synthesize each undecamer was 1 to 2 weeks. Optimization of the steps to achieve this is described in detail.

Autoradiography↗