General introduction and thesis outline 17 1 1.2.2 THE CLINICAL OUTCOMES OF HRHPV INFECTIONS The various outcomes of exposure of cervical epithelial cells to hrHPV are a transient infection, a productive infection and a transforming infection 25. Around 80% of the genital HPV infections are transient, cleared within months following infection and do not result in cervical pathology. Most of the remaining hrHPV infections are productive infections. Productive infections are characterised by the release of new viral particles through shedding of terminally differentiated cells in the upper layer of the epithelium. The viral life cycle relies heavily on the differentiation process of the infected squamous epithelium. The viral gene expression is regulated by multiple promoters and complex mRNA splicing patterns that enable the expression of different proteins at different stages of the viral life cycle 44, 45. A productive infection with hrHPV begins when virions enter basal keratinocytes of differentiating cervical epithelium through defects in the epithelial covering 46. Viral entry requires binding of virions to heparin sulphate proteoglycans (HSPGs) on the cell surface, resulting in a conformational change, followed by site-specific enzymatic cleavage of the L2 protein by furin, a cell-encoded proprotein convertase 47, 48. The conformational change resulting from the proteolytic cleavage facilitates virus internalisation. The infected basal keratinocytes or stem cells become a reservoir of infection, where the viral genome enters the cell nucleus and the episome is maintained at low copy numbers and expressed at low levels. Infected daughter cells are produced as the cells divide and migrate towards the epithelial surface, where different viral proteins are expressed at different stages of epithelial differentiation (Figure 1.4). In the lower layers of the epithelium, viral proteins E6 and E7 activate the cell cycle machinery to accomplish HPV replication and drive cells through cell cycle and stimulate cell division (cycling cells marked with red nuclei). In the middle layers, viral genome amplification is enhanced by the combined action of viral proteins E1, E2, E6 and E7. Indirectly, the amplification success is dependent on the modifying effect of E4 and E5 on the cellular environment in the S or G2 phases of the cell cycle (presence of E4 is marked in purple, with red nuclei indicating replication competence). In the upper layers, the productive viral life-cycle is completed, as the cells leave the cell cycle. Viral proteins L1 and L2 are produced in a subset of the E4-positive cells, allowing the amplified viral genomes to be packaged and released as viral particles 49. Productive hrHPV infections may give rise to mild to moderate cellular abnormalities, which are histologically recognisable as CIN1 or CIN2 lesions. These so-called productive CIN lesions tend to regress spontaneously within 1-2 years 25. The expression of the HPV E4 protein during the final stage of the viral life cycle is a marker of productive
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