Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator

Abstract

In order to ensure a productive life cycle, human papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins E6 and E7 results in the immortalization of the infected epithelial cells and thus prevents the production of mature virions. Ectopically expressed E2 has been shown to suppress transcription of the HPV E6 and E7 region in cell lines where the viral DNA is integrated into the host genome, resulting in growth inhibition. However, it has been demonstrated that growth control of these cell lines can also occur independently of HPV E2 transcriptional activity in high-risk HPV types. In addition, E2 is unable to suppress transcription of the same region in cell lines derived from cervical tumors that harbor only episomal copies of the viral DNA. Here we show that HPV type 16 (HPV-16) E2 is capable of inhibiting HPV-16 E7 cooperation with an activated ras oncogene in the transformation of primary rodent cells. Furthermore, we demonstrate a direct interaction between the E2 and E7 proteins which requires the hinge region of E2 and the zinc-binding domain of E7. These viral proteins interact in vivo, and E2 has a marked effect upon both the stability of E7 and its cellular location, where it is responsible for recruiting E7 onto mitotic chromosomes at the later stages of mitosis. These results demonstrate a direct role for E2 in regulating the function of E7 and suggest an important role for E2 in directing E7 localization during mitosis.Human papillomaviruses (HPVs) are a large family of small, double-stranded DNA viruses. They infect cutaneous and mucosal epithelial tissue at different anatomical locations, resulting in a variety of clinical symptoms ranging from benign warts to invasive genital cancers (13,29). Infection with the high-risk types, most commonly HPV type 16 (HPV-16) and HPV-18, has been associated with the development of more than 99% of cervical cancer cases. The tumorigenicity of HPV is dependent on the activity of two virally encoded oncoproteins, E6 and E7. These bind at a high affinity and disrupt the function of p53 (46) and the retinoblastoma tumor suppressor protein pRB (6, 20), respectively. While E6 plays a role in inhibiting apoptosis and interfering with cell adhesion and polarity (30), E7 acts by driving S-phase progression, regulating gene expression, and interfering with the activities of cyclins and cyclin-dependent kinases (23). In addition to inactivating the function of pRB, other cellular targets of E7 include the TATA box-binding protein (TBP) (31), TBP-associated factors (35), members of the AP-1 transcription factor family (2), and histone deacetylases (7). E7 is a small phosphoprotein that shares some sequence homology with the adenovirus E1a protein and the simian virus 40 large T antigen (8). E7 is 98 amino acids in length and contains a zinc-binding domain in the C-terminal region whose structural integrity is necessary for the activity of E7 (2, 26). While both E6 and E7 cooperate to induce immortalization o...

Inhibition of HPV-16 E7 oncogenic activity by HPV-16 E2

“…After 48 h, the levels of pRb protein were ascertained by western blotting and the results obtained are shown in Figure 3a. As can be seen, E7-induced degradation of pRb was rescued in the presence of E2, even though the expression level of E7 was augmented in the presence of E2 (Figure 3b), as reported previously (Gammoh et al, 2006). These results demonstrate that E2 can inhibit E7-induced degradation of pRb and provide a molecular explanation for the ability of E2 to inhibit transforming activity of E7 in BRK cells (Gammoh et al, 2006).…”

“…Previously, we presented evidence that the activity of E7 can be controlled through a direct interaction with E2 (Gammoh et al, 2006), resulting in an inhibition of transforming activity of E7. In addition, E2 also increased the stability of the E7 protein and induced its re-localization to mitotic chromosomes.…”

“…Previous studies had shown that E7 induction of centrosome duplication defects could be monitored in a transient transfection assay in U2OS cells (Duensing et al, 2000). To analyse the effects of E2 on this, centrosome numbers were monitored in U2OS cells transiently transfected with a hemagglutinin (HA)-tagged HPV-16 E7 expression plasmid, in the presence or absence of an HPV-16 E2 expression plasmid (Gammoh et al, 2006). At 24 h after transfection, cells were treated with nocodazole for a further 24 h to induce a G 2 /M-phase arrest, in which centrosome duplication would be expected to have been completed.…”

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

“…The viral E6 promoter might be down-regulated by increasing levels of viral E2 protein expressed from the increasing viral DNA templates (Chow and Broker 2007 and references therein). Alternatively, elevated E2 protein has been reported to directly bind and inactivate E7 (Gammoh et al 2006). …”

The Role of E2 Proteins in Papillomavirus DNA Replication

“…E2-dependent activities are also modulated by interactions with Brm, a chromatin remodeling protein associated with SWI/SNF ATP-dependent chromatin remodeling complex, and with EP400, a component of the NuA4/TIP60 histone acetylase complex, and SMCX, also known as histone demethylase JARID1C , and Tax1BP1 (Kumar et al, 2007;Smith et al, 2010;Wang et al, 2009b). E2 binds to the papillomavirus oncoproteins E6 and E7, leading to the modulation of their functions (Gammoh et al, 2006;Grm et al, 2005). E2 has activities that inhibit cell growth.…”