Biomedical subjects

T L McClowry

Publications and source records attributed to T L McClowry.

3 recordsLinked to original sources

PubMed1999

Nucleotide sequence and characterization of human papillomavirus type 83, a novel genital papillomavirus.

Studies of human papillomaviruses (HPV) are hampered by the lack of a conventional culture system, because HPV completes its life cycle only in fully differentiated human tissue. To overcome this obstacle, the athymic mouse xenograft system has been used to study the pathogenesis of a limited number of HPV types. We recently reported the propagation of a novel HPV type in the mouse xenograft system and the cloning of its genome. Consensus primer PCR had previously identified this virus as MM7, LVX82, or PAP291. Here we report the nucleotide sequence of the 8104-bp genome of this virus, now called HPV 83. HPV 83 is most closely related to HPV 61 and HPV 72, placing it in the papillomavirus genome homology group A3. Based on limited epidemiological data, the histological appearance of infected human foreskin implants, and the structure of the predicted HPV 83 E7 protein, this virus is probably of at least intermediate cancer risk. Like other papillomaviruses, HPV 83 produces an E1 E4, E5 transcript, but the position of the splice acceptor differs from that of other HPVs. The presence of an E5 open reading frame in the HPV 83 genome is uncertain; the most likely candidate to be the HPV 83 E5 protein has some structural similarity to the bovine papillomavirus 1 E5 oncoprotein, and is unlike most other HPV E5 proteins. HPV 83 is a relatively prevalent genital papillomavirus that has the largest genome of any characterized HPV and several other novel structural features that merit further study.

Amino Acid Sequence↗

PubMed1998

A human papillomavirus related to human papillomavirus MM7/LVX82 produces distinct histological abnormalities in human foreskin implants grown as athymic mouse xenografts.

Studies of human papillomaviruses (HPVs) are hampered by the lack of a conventional culture system because HPV completes its life cycle only in fully differentiated human tissue. To overcome this obstacle, the athymic mouse xenograft system has been used to study the pathogenesis of HPV 11 and to develop neutralizing assays for vaccine development. Recently, HPV 40 has been produced in this system, and HPV 16 has been produced using mice with severe combined immune deficiency. To identify and characterize additional genital HPV types for similar studies, condylomata acuminata lesions containing a high copy number of HPV and detectable L1 major capsid protein were used to prepare infectious virus stocks. Human foreskin fragments were infected with the virus preparations and implanted under the renal capsules of athymic mice. After 5 months of growth, implant tissue was removed and processed for studies to detect HPV infection. Evidence of HPV infection was noted in some of the implants, but in contrast to HPV 11-infected epithelium, the implants derived from the new virus preparations contained a lesser degree of acanthosis, less developed koilocytosis, and a reduced number of preserved nuclei in the hyperkeratotic material within the cyst lining. The L1 consensus region was amplified by polymerase chain reaction from implant DNA and sequenced. Alignment of the amplified sequences with those in the HPV sequence database showed that the 452-bp amplimer was closely related but not identical to HPV LVX82 and HPV MM7 (also called Pap 291). The entire 7.9-kb genome was amplified by polymerase chain reaction and cloned. The presence of virions of the new isolate (named HPV IU) in the implants was verified by immunohistochemical detection of L1 major capsid protein and by demonstration of virion particles by electron microscopy. A second extract was made from one of the new implants and used to successfully propagate HPV IU. These experiments demonstrate that experimental infection of human epithelium with the new isolate, HPV IU, is associated with histological abnormalities that differ in potentially important ways from the changes observed in experimental HPV 11 infection.

Animals↗

PubMed1998

Expression of the human papillomavirus type 11 E5A protein from the E1E4,E5 transcript.

The abundant human papillomavirus type 11 (HPV 11) E1E4,E5 transcript potentially encodes the E1E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3' end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1E4,E5a construct that lacked the 5' noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1wedgeE4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1E4,E5 transcript is a functional message that can support both E1E4 and E5a expression in eukaryotic cells.

Animals↗