The Reservoir of Persistent Human Papillomavirus Infection; Strategies for Elimination Using Anti-Viral Therapies.

Human Papillomaviruses have co-evolved with their human host, with each of the over 200 known HPV types infecting distinct epithelial niches to cause diverse disease pathologies. Despite the success of prophylactic vaccines in preventing high-risk HPV infection, the development of HPV anti-viral therapies has been hampered by the lack of enzymatic viral functions, and by difficulties in translating the results of in vitro experiments into clinically useful treatment regimes. In this review, we discuss recent advances in anti-HPV drug development, and highlight the importance of understanding persistent HPV infections for future anti-viral design. In the infected epithelial basal layer, HPV genomes are maintained at a very low copy number, with only limited viral gene expression; factors which allow them to hide from the host immune system. However, HPV gene expression confers an elevated proliferative potential, a delayed commitment to differentiation, and preferential persistence of the infected cell in the epithelial basal layer, when compared to their uninfected neighbours. To a large extent, this is driven by the viral E6 protein, which functions in the HPV life cycle as a modulator of epithelial homeostasis. By targeting HPV gene products involved in the maintenance of the viral reservoir, there appears to be new opportunities for the control or elimination of chronic HPV infections.

Role of E6 in Maintaining the Basal Cell Reservoir during Productive Papillomavirus Infection.

Papillomaviruses exclusively infect stratified epithelial tissues and cause chronic infections. To achieve this, infected cells must remain in the epithelial basal layer alongside their uninfected neighbors for years or even decades. To examine how papillomaviruses achieve this, we used the in vivo MmuPV1 (Mus musculus papillomavirus 1) model of lesion formation and persistence. During early lesion formation, an increased cell density in the basal layer, as well as a delay in the infected cells' commitment to differentiation, was apparent in cells expressing MmuPV1 E6/E7 RNA. Using cell culture models, keratinocytes exogenously expressing MmuPV1 E6, but not E7, recapitulated this delay in differentiation postconfluence and also grew to a significantly higher density. Cell competition assays further showed that MmuPV1 E6 expression led to a preferential persistence of the cell in the first layer, with control cells accumulating almost exclusively in the second layer. Interestingly, the disruption of MmuPV1 E6 binding to MAML1 protein abrogated these phenotypes. This suggests that the interaction between MAML1 and E6 is necessary for the lower (basal)-layer persistence of MmuPV1 E6-expressing cells. Our results indicate a role for E6 in lesion establishment by facilitating the persistence of infected cells in the epithelial basal layer, a mechanism that is most likely shared by other papillomavirus types. Interruption of this interaction is predicted to impede persistent papillomavirus infection and consequently provides a novel treatment target. IMPORTANCE Persistent infection with high-risk HPV types can lead to development of HPV-associated cancers, and persistent low-risk HPV infection causes problematic diseases, such as recurrent respiratory papillomatosis. The management and treatment of these conditions pose a considerable economic burden. Maintaining a reservoir of infected cells in the basal layer of the epithelium is critical for the persistence of infection in the host, and our studies using the mouse papillomavirus model suggest that E6 gene expression leads to the preferential persistence of epithelial cells in the lower layers during stratification. The E6 interaction with MAML1, a component of the Notch pathway, is required for this phenotype and is linked to E6 effects on cell density and differentiation. These observations are likely to reflect a common E6 role that is preserved among papillomaviruses and provide us with a novel therapeutic target for the treatment of recalcitrant lesions.

Principles of epithelial homeostasis control during persistent human papillomavirus infection and its deregulation at the cervical transformation zone.

Human papillomaviruses establish a reservoir of infection in the epithelial basal layer. To do this they limit their gene expression to avoid immune detection and modulate epithelial homeostasis pathways to inhibit the timing of basal cell delamination and differentiation to favour persistence. For low-risk Alpha papillomaviruses, which cause benign self-limiting disease in immunocompetent individuals, it appears that cell competition at the lesion edge restricts expansion. These lesions may be considered as self-regulating homeostatic structures, with epithelial cells of the hair follicles and sweat glands, which are proposed targets of the Beta and Mu papillomaviruses, showing similar restrictions to their expansion across the epithelium as a whole. In the absence of immune control, which facilitates deregulated viral gene expression, such lesions can expand, leading to problematic papillomatosis in afflicted individuals. By contrast, he high-risk Alpha HPV types can undergo deregulated viral gene expression in immunocompetent hosts at a number of body sites, including the cervical transformation zone (TZ) where they can drive the formation of neoplasia. Homeostasis at the TZ is poorly understood, but involves two adjacent epithelial cell population, one of which has the potential to stratify and to produce a multilayed squamous epithelium. This process of metaplasia involves a specialised cell type known as the reserve cell, which has for several decades been considered as the cell of origin of cervical cancer. It is becoming clear that during evolution, HPV gene products have acquired functions directly linked to their requirements to modify the normal processes of epithelial homestasis at their various sites of infection. These protein functions are beginning to provide new insight into homeostasis regulation at different body sites, and are likely to be central to our understanding of HPV epithelial tropisms.

Mutations in HPV18 E1

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation.

HPV16 and 18 genome amplification show different E4-dependence, with 16E4 enhancing E1 nuclear accumulation and replicative efficiency via its cell cycle arrest and kinase activation functions.

To clarify E1^E4's role during high-risk HPV infection, the E4 proteins of HPV16 and 18 were compared side by side using an isogenic keratinocyte differentiation model. While no effect on cell proliferation or viral genome copy number was observed during the early phase of either virus life cycle, time-course experiments showed that viral genome amplification and L1 expression were differently affected upon differentiation, with HPV16 showing a much clearer E4 dependency. Although E4 loss never completely abolished genome amplification, its more obvious contribution in HPV16 focused our efforts on 16E4. As previously suggested, in the context of the virus life cycle, 16E4s G2-arrest capability was found to contribute to both genome amplification success and L1 accumulation. Loss of 16E4 also lead to a reduced maintenance of ERK, JNK and p38MAPK activity throughout the genome amplifying cell layers, with 16E4 (but not 18E4) co-localizing precisely with activated cytoplasmic JNK in both wild type raft tissue, and HPV16-induced patient biopsy tissue. When 16E1 was co-expressed with E4, as occurs during genome amplification in vivo, the E1 replication helicase accumulated preferentially in the nucleus, and in transient replication assays, E4 stimulated viral genome amplification. Interestingly, a 16E1 mutant deficient in its regulatory phosphorylation sites no longer accumulated in the nucleus following E4 co-expression. E4-mediated stabilisation of 16E2 was also apparent, with E2 levels declining in organotypic raft culture when 16E4 was absent. These results suggest that 16E4-mediated enhancement of genome amplification involves its cell cycle inhibition and cellular kinase activation functions, with E4 modifying the activity and function of viral replication proteins including E1. These activities of 16E4, and the different kinase patterns seen here with HPV18, 31 and 45, may reflect natural differences in the biology and tropisms of these viruses, as well as differences in E4 function.

Characterization of beta papillomavirus E4 expression in tumours from Epidermodysplasia Verruciformis patients and in experimental models.

This study provides a first characterisation of ß-HPV life-cycle events in tumours abscised from EV patients (the human model of ß-HPV-induced skin cancer), and shows how changes in E4 expression patterns relate to disease severity. ß-HPV life-cycle has also been reconstructed in organotypic raft cultures created using EV-derived keratinocytes. In EV lesions and raft cultures, abundant cytoplasmic E4 expression was detectable in differentiating cells along with viral genome amplification as reported for other HPV types. E4 expression was also seen in PCNA-positive basal cells in some EV skin cancers as well as in tumours from HPV8CER (Complete Early Region) transgenic mice. In these lesions, E4 staining extended throughout the full thickness of the epithelium and was apparent in the markedly atypical cells. The loss of such staining at the tumour border suggests a distinct type of E4 dysregulation that may be exploited as a marker of viral expression during ß-HPV-associated skin cancer progression.

E1--E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium.

The keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells. The human papilloma virus (HPV) type 16 E1--E4 protein accumulates in the upper layers of HPV16-infected epithelium and is known to associate with and reorganise the keratin IF network in cells in culture. Here, we show that this function is conserved amongst a number of HPV alpha-group E1--E4 proteins and that the differentiation-dependent keratins are also targeted. Using time-lapse microscopy, HPV16 E1--E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. Network disruption was accompanied by keratin hyperphosphorylation at several sites, including K8 S73, which is typically phosphorylated in response to stress stimuli. Keratin immunoprecipitated from E1--E4-expressing cells was also found to be ubiquitylated, indicating that it is targeted for proteasomal degradation. Interestingly, the accumulation of hyperphosphorylated, ubiquitylated E1--E4-keratin structures was found to result in an impairment of proteasomal function. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1--E4 and provide an insight into the depletion of keratin co-incident with E1--E4 accumulation observed in HPV-infected epithelium.

Structural analysis reveals an amyloid form of the human papillomavirus type 16 E1--E4 protein and provides a molecular basis for its accumulation.

The abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1--E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1--E4 into amyloid-like fibrils, which bind to thioflavin T. The C-terminal region is highly amyloidogenic, and its deletion abolishes amyloid staining and prevents E1--E4 accumulation. Amyloid-imaging probes can detect 16E1--E4 in biopsy material, as well as 18E1--E4 and 33E1--E4 in monolayer cells, indicating structural conservation. Our results suggest a role for fibril formation in facilitating the accumulation of E1--E4 during HPV infection.