Explore the vast range of titles available.

In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the U L / b ′ region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV.

HCMV encodes a plethora of immune-modulating functions, many of which have yet to be assigned to specific genes. In prospect of performing high-throughput screens to identify and characterise such functions, a library of recombinant adenoviruses (RAds) each encoding a V5 epitope-tagged HCMV protein was generated. Protein expression was validated and characterised for the vast majority of RAds by western blot and immunofluorescence. HCMV has been reported to both upregulate cell surface expression of Fas, and render cells resistant to Fas-mediated killing. This thesis demonstrated that Fas levels are markedly reduced at the surface of HCMV-infected cells as an early function that persists through the late phase. Screening a panel of HCMV deletion mutants eliminated 83 genes as not required for Fas downregulation, while screening the RAd library did not identify any single HCMV gene as being sufficient for this function. Deep sequencing of the HCMV transcriptome recently led to the identification of UL150A as a novel protein-coding gene. To test this prediction, UL150A was tagged within the strain Merlin genome. UL150A was shown to encode multiple protein products, and be expressed with early and late kinetics. In a screen of the RAd library, gpUL4 was observed to be secreted from cells. To investigate this function in the context of HCMV infection, an epitope-tag was inserted at the 3’-end of the UL4 gene in the strain Merlin genome. Tagged gpUL4 was secreted from cells infected with strain Merlin. Secreted gpUL4 was more heavily glycosylated, and produced in greater abundance than its intracellular counterpart late in infection. Active secretion would be consistent with gpUL4 acting as a virokine, cytokine or cytokine/chemokine-binding protein. gpUL4 purified from supernatants of Merlin- or RAd-UL4-infected cells inhibited NK cell degranulation. Furthermore, gpUL4 did not copurify with virus particles, indicating it is unlikely to be a virion component.