Investigation of Protein Products Encoded by the Human Cytomegalovirus US22 Family Genes UL23, UL24, UL43 and US22

Adair, Richard (2002) Investigation of Protein Products Encoded by the Human Cytomegalovirus US22 Family Genes UL23, UL24, UL43 and US22. PhD thesis, University of Glasgow.

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This thesis reports the investigation of four Human Cytomegalovirus (HCMV) genes (UL23, UL24, UL43 and US22) which belong to the twelve-member HCMV US22 gene family. In order to investigate whether pUL23, pUL24, pUL43 and pUS22 were structural or non-structural proteins, HCMV (strain AD169) particles were purified from the culture medium of infected HFFF-2 cells. Three types of particles were resolved in glycerol potassium/tartrate gradients - virions, dense bodies and non-infectious enveloped particles (NIEPs). Western immunoblotting showed that pUL24 and pUL43 were present in all particle types. pUL23 and pUS22 were present in low amounts in virions and dense bodies, and although not investigated, due to low particle numbers, are expected to be present in NIEPs. To confirm that these were virus particle components and not simply associated with co-purifying cell debris, purified virus particles were examined in the electron microscope (EM). The presence of pUL23, pUL24, pUL43 and pUS22 in virus particles was confirmed by immuno-gold tagging of the tegument region of virions and the matrix component of dense bodies. The envelope was removed from purified virus particles by treatment with detergent and pUL23, pUL24, pUL43 and pUS22 each separated with the capsid/tegument fraction, confirming their status as HCMV tegument proteins. The intracellular location of pUL24, pUL43 and pUS22 in HCMV infected HFFF-2 cells was investigated by immunofluorescence. pUS22 was confirmed to be present in the nucleus throughout infection. In contrast, pUL24 and pUL43 were non-nuclear proteins that accumulated in the perinuclear region, concentrating in a structure juxtaposed to the nucleus. The intracellular distribution of the four proteins was further investigated at the level of cell ultra-structure by immuno-gold thin section EM. With the exception of their presence in cytoplasmic virus particles pUL23, pUL24 and pUL43 were exclusively located in large cytoplasmic protein aggregates, which correlate with the juxtanuclear structure visualised by immunofluoresence. pUS22 on the other hand appeared to be distributed throughout the cell. The protein aggregates manifested in two forms: complex structures which appeared to lack a boundary membrane and smaller, membrane-bound aggregates resembling dense bodies. It has been suggested that the juxtanuclear structure is a site of HCMV tegument acquisition and particle maturation, and the findings reported here are in agreement with this proposition. The pUL23, pUL24 and pUL43 tegument proteins could only have been acquired by particles interacting with the protein aggregates and indeed tegumented, but non-enveloped, particles were frequently associated with the complex-type protein aggregates. While the HCMV UL43 gene is known to be non-essential, it was not known whether or not the UL23 and UL24 genes were required for virus replication in vitro. In order to isolate and propagate knockout mutant viruses in essential genes it is necessary to produce complementing cell lines. Retinal pigmented epithelial cells (RPE) and HFF cells immortalised due to expression of either human telomerase reverse transcriptase (hTERT) or the Human Papillomavirus (HPV) type 16 E6/E7 transforming genes, respectively, were employed to construct cell lines expressing pUL23, pUL24 and pUL43. Several cloned hTERT-RPE cell lines constitutively and stably expressing pUL43 were successfully obtained. However, irrespective of the cell line used, no pUL23 or pUL24 expressing line was obtained. In order to investigate the function provided by the HCMV UL23 and UL24 genes attempts were made to generate UL23 and UL24 deletion mutants. Precise deletions in the UL23 or UL24 open reading frame (ORF) were engineered and replaced with the E. co/i guanosine phosphoribosyl transferase (gpt) gene, to serve as a selectable marker gene. However, after four rounds of virus replication in the presence of mycophenolic acid and xanthine to enrich for gpt containing virus, followed by four rounds of plaque picking, all of the plaques yielded the wild type DNA fragment profile in Southern blots. Inability to isolate a UL23 or UL24 deletion mutant was probably due to the poor efficiency of transfection of HFFF-2 cells and/or to a low level of recombination between the HCMV plasmid mutation and the wild type virus genome during the initial stages of the experiment. The most important finding from this investigation was the demonstration that pUL23, pUL24, pUL43 and pUS22 are tegument proteins. Three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components. Thus, at least seven of the twelve US22 family genes code for tegument proteins, suggesting that the products of the remaining five might be similarly located. This demonstrates for the first time a common biological feature among the US22 family members.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Derrick Dargan
Keywords: Virology, Genetics, Molecular biology
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-76269
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:12
Last Modified: 19 Nov 2019 16:12

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