Biomedical subjects

Keith N Leppard

Publications and source records attributed to Keith N Leppard.

6 recordsLinked to original sources

PubMed2006

Interaction of the adenovirus type 5 E4 Orf3 protein with promyelocytic leukemia protein isoform II is required for ND10 disruption.

Nuclear domain 10 (ND10s), or promyelocytic leukemia protein (PML) nuclear bodies, are spherical nuclear structures that require PML proteins for their formation. Many viruses target these structures during infection. The E4 Orf3 protein of adenovirus 5 (Ad5) rearranges ND10s, causing PML to colocalize with Orf3 in nuclear tracks or fibers. There are six different PML isoforms (I to VI) present at ND10s, all sharing a common N terminus but with structural differences at their C termini. In this study, PML II was the only one of these six isoforms that was found to interact directly and specifically with Ad5 E4 Orf3 in vitro and in vivo; these results define a new Orf3 activity. Three of a series of 18 mutant Orf3 proteins were unable to interact with PML II; these were also unable to cause ND10 rearrangement. Moreover, in PML-null cells that contained neoformed ND10s comprising a single PML isoform, only ND10s formed of PML II were rearranged by Orf3. These data show that the interaction between Orf3 and PML II is necessary for ND10 rearrangement to occur. Finally, Orf3 was shown to self-associate in vitro. This activity was absent in mutant Orf3 proteins that were unable to form tracks and to bind PML II. Thus, Orf3 oligomerization may mediate the formation of nuclear tracks in vivo and may also be important for PML II binding.

Adenovirus E4 Proteins↗

PubMed2005

Isoforms of the promyelocytic leukemia protein differ in their effects on ND10 organization.

The PML protein is a defining constituent of subnuclear structures known as ND10. PML is expressed as a series of primary sequence isoforms through alternative RNA processing. Expression of each of six distinct PML isoforms that differed in their C-terminal domains caused reproducible differences in the number, size, and shape of ND10 in both transformed cell lines and diploid fibroblasts. In each case, PML from the endogenous genes was reorganized to participate with the exogenously expressed PML in the new configuration of ND10. Variation in ND10 number is known to occur during the cell cycle; however, the cell cycle distribution of the transfected cells that displayed these altered ND10 was similar for all six PML isoforms. Given our findings, the precise level of expression of the different PML isoforms under particular physiological conditions will be an important determinant of ND10 organization and function and is a potential point of regulation of PML/ND10 function.

Amino Acid Sequence↗

PubMed2004

HPV E6 proteins interact with specific PML isoforms and allow distinctions to be made between different POD structures.

Mucosal human papillomaviruses (HPVs) are the causative agents of a number of human pathologies, including benign condylomas, as well as of the majority of cervical cancers and their high-grade precursor lesions. Although the viral E6 protein is known to be essential for driving malignant progression of HPV-infected cells, there are still many uncertainties about its mode of action. In this study, we have analysed the intracellular distribution of the E6 oncoproteins from the high-risk HPV-18 and the low-risk HPV-11. We show that both E6 proteins localize within the nucleus in nuclear bodies that are confocal with the promyelocytic leukaemia (PML) protein. Using a panel of different PML isoforms, we demonstrate specific co-localization between the E6 proteins and PML isoforms I-IV, but not with PML isoforms V and VI. We also demonstrate the interaction between E6 and a subset of PML isoforms in vivo. As a consequence of this interaction, the insoluble form of PML IV is destabilized by HPV-18 E6 through a proteasome-dependent pathway. Interestingly, both HPV-11 E6 and HPV-18 E6 can readily overcome PML IV-induced cellular senescence in primary cells. These results show separable functions for different PML isoforms that are specifically targeted by the HPV E6 oncoproteins.

Alternative Splicing↗

PubMed2004

Activation of the early-late switch in adenovirus type 5 major late transcription unit expression by L4 gene products.

The adenovirus major late transcription unit (MLTU) encodes multiple proteins from five regions, L1 to L5, through differential splicing and polyadenylation. MLTU expression is temporally regulated; only a single product from L1 (52/55K) is expressed prior to replication, but a subsequent switch, the mechanism of which has not been defined, leads to full expression that encompasses L1 IIIa and all L2 to L5 products. Transfection of a plasmid containing the complete MLTU gave a full array of proteins in proportions similar to those in a late infection, and in a time course, the temporal pattern of expression in a natural infection was reproduced. However, a plasmid truncated after the L3 poly(A) site exclusively expressed the L1 52/55K protein and was defective in the switch to full gene expression from L1 to L3. The L4 33K protein, supplied in trans, was sufficient to upregulate cytoplasmic mRNA for MLTU products characteristic of the late pattern of expression to levels comparable to those produced by the full-length MLTU. There was a corresponding increase in expression of the L1 IIIa, L2, and L3 proteins, except hexon. Hexon protein expression additionally required both the L4 100K protein in trans and sequences downstream of the L3 poly(A) site in cis. These results indicate that induction of L4 protein expression is a key event in the early-late switch in MLTU expression, which we propose is precipitated by small amounts of L4 expression in a feed-forward activation mechanism.

Adenoviruses, Human↗

PubMed2004

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

Adenoviridae↗

PubMed2003

Nuclear matrix localization and SUMO-1 modification of adenovirus type 5 E1b 55K protein are controlled by E4 Orf6 protein.

Human adenovirus serotype 5 encodes three proteins, E1b 55K, E4 Orf3 and E4 Orf6, which interact with each other and with components of the nucleus to regulate mRNA processing and export, viral DNA replication and p53-dependent apoptosis. Previous studies have shown that, during wild-type infection, 55K associates initially with structures termed ND10, which are sites of localization of the promyelocytic leukaemia protein, and then moves, dependent upon its interaction with Orf6, to the establishing virus replication centres. Absence of either Orf3 or Orf6 affects the localization of 55K and so may affect its function. In this study, the influence of Orf3 and Orf6 expression on the association of 55K with the insoluble matrix fraction of the nucleus and with ND10 particularly was examined. Overexpression of Orf6 was sufficient to block the association of 55K with this fraction, irrespective of the presence of Orf3. This effect depended upon the two proteins being able to interact. However, the association of 55K with ND10, which persists throughout infection in the absence of Orf6, required Orf3 to be present, thus distinguishing two subsets of matrix-associated 55K. A modified form of 55K, formation of which was blocked by mutating the known site of SUMO-1 attachment, was more abundant in the absence of Orf6 but unaffected by the absence of Orf3. Thus, this modification is favoured when 55K remains associated with the matrix but does not correlate with its stable association with ND10, many components of which are modified by SUMO-1.

Adenovirus E1B Proteins↗