Risk factors associated with oral Human Papillomavirus (HPV) prevalence within a young adult population.

BACKGROUND: The prevalence of, and risk factors for, genital Human Papillomavirus (HPV) infections within the young adult population are well-established; the same is not known for oral HPV. This observational study aimed to determine oral HPV prevalence and abundance within a UK young adult population, and examine if sexual practices and established risk factors of oropharyngeal squamous cell carcinomas (OPSCCs) (such as smoking and alcohol consumption) influenced HPV prevalence. METHODS: Convenience sampling was used to recruit a small sample of 452 UK-based young adults studying at a higher education (HE) institution to the study; the study was not powered. A highly sensitive real-time PCR HPV screening method was developed for the detection of multiple HPV subtypes from oral swabs. HPV-positive samples were subsequently screened by qPCR for viral subtypes HPV-6, HPV-11, HPV-16, HPV-18. Results were analysed by univariate and multivariate methods and stratified for gender, with lifestyle behaviour data collected via questionnaire. Socio-economic status was not captured within the questionnaire. RESULTS: We found a high oral HPV prevalence of 22.79%, with a dominance of high-risk viral type HPV-16 (prevalence 19.12%; abundance average 1.08 × 105 copies/million cells) detected within healthy young adults. Frequent smoking (p = .05), masturbation (p = .029), and engagement in multiple sexual activities (p = .057), were found to be associated with oral HPV prevalence, and HPV-16 prevalence, whilst behaviours traditionally associated with genital HPV were not. CONCLUSIONS: Our results strengthen the link between sexual practices and oral HPV transmission. We suggest that young adults should be considered high-risk for the contraction of oral HPV, although acknowledge that this sample of HE students may not be representative of the wider population. We show that high-risk HPV-16 is prevalent in the healthy population, as well as dominating within OPSCC; this study is one of the first to determine the dominance of oral HPV-16 prevalence and abundance within this population, presenting a clear need for greater awareness of oral HPV infections, and the risk factors for HPV-positive OPSCC within young adults.

The role of protein kinase A regulation of the E6 PDZ-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18.

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1

The G2/M arrest function of human papillomavirus (HPV) E4 proteins is hypothesized to be necessary for viral genome amplification. Full-length HPV18 E1^E4 protein is essential for efficient viral genome amplification. Here we identify key determinants within a CDK-bipartite consensus recognition motif in HPV18 E1^E4 that are critical for association with active CDK-cyclin complexes and in vitro phosphorylation at the predicted CDK phosphorylation site (threonine 23). The optimal cyclin-binding sequence ((43)RRLL(46)) within this E4 motif is required for G2/M arrest of primary keratinocytes and correlates with cytoplasmic retention of cyclin B1, but not cyclin A. Disruption of this motif in the E4 ORF of HPV18 genomes, and the subsequent generation of stable cell lines in primary keratinocytes revealed that this motif was not essential for viral genome amplification or L1 capsid protein induction. We conclude that the HPV18 E4 G2/M arrest function does not play a role in early vegetative events.

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells.

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

The full-length E1E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression.

Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1E4 protein. To determine the role of E1E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1E4 expression, and to HPV16 where only C-terminal truncations in E1E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1E4 proteins.

Role for Wee1 in inhibition of G2-to-M transition through the cooperation of distinct human papillomavirus type 1 E4 proteins.

The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1;E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1;E4 sequences. HPV1 E4 inhibits G(2)-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1;E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr(15) phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G(2), indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G(2) block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G(2)-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G(2) arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G(2)-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.

Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis.

Posttranslational modification-oligomerization, phosphorylation, and proteolytic cleavage-of the human papillomavirus (HPV) E4 protein occurs as the infected keratinocytes migrate up through the suprabasal wart layers. It has been postulated that these events modify E4 function during the virus life cycle. In HPV type 1 (HPV1)-induced warts, N-terminal sequences are progressively cleaved from the full-length E4 protein (E1(wedge)E4) of 17 kDa to produce a series of polypeptides of 16, 11 and 10 kDa. Here, we have shown that in human keratinocytes, a truncated protein (E4-16K), equivalent to the 16-kDa species, mediated a G(2) arrest in the cell cycle that was dependent on a threonine amino acid in a proline-rich domain of the protein. Reconstitution of cyclin B1 expression in E4-16K cells reversed the G(2) arrest. Expression of E4-16K also induced chromosomal rereplication, and this was associated with aberrant nuclear morphology. Perturbation of the mitotic cell cycle was a biological activity specific to the truncated protein. However, coexpression of the full-length E1(wedge)E4 protein and the truncated E4-16K protein inhibited normal cellular proliferation and cellular DNA rereplication but did not prevent cells from arresting in G(2). Our findings provide the first evidence to support the hypothesis that proteolytic cleavage of the E1(wedge)E4 protein modifies its function. Also, different forms of the HPV1 E4 protein cooperate to negatively influence keratinocyte proliferation. We predict that these distinct biological activities of E4 act to support efficient amplification of the viral genome in suprabasal keratinocytes.

The ND10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 E4 intranuclear inclusion bodies in cultured keratinocytes and in warts.

Human papillomavirus type 1 (HPV1) E4 protein is associated with cytoplasmic and nuclear inclusions in productively infected keratinocytes. Here we have used transient expression of HPV1 E4 (also known as E1E4) protein in keratinocytes to reproduce formation of E4 inclusions. Immunofluorescence analysis showed that progressive formation of inclusions correlated with diminished colocalization between E4 and keratin intermediate filaments (IFs). Our results support a model in which the HPV1 E4-keratin IF association is transient, occurring only at an early stage of inclusion formation. We also demonstrate that E4 induces relocation of the promyelocytic leukemia protein (PML) from multiple intranuclear speckles (ND10 bodies) to the periphery of nuclear E4 inclusions and that this activity is specific to full-length E4 protein. Analysis of HPV1-induced warts demonstrated that nuclear PML-E4 inclusions were present in productively infected keratinocytes, indicating that reorganization of PML occurs during the virus's replication cycle. It has been suggested that ND10 bodies are the sites for papillomavirus genome replication and virion assembly. Our finding that E4 induces reorganization of ND10 bodies in vitro and in vivo is further strong evidence that these domains play an important role in the papillomavirus life cycle. This study indicates that HPV1 is analogous to other DNA viruses that disrupt or reorganize ND10 domains, possibly to increase efficiency of virus infection. We hypothesize that HPV1 E4-induced reorganization of PML is necessary for efficient replication of the virus during the virus-producing phase.