Human papillomavirus (HPV) E7 induces prolonged G2 following S phase reentry in differentiated human keratinocytes

Abstract

The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that HPV-18 DNA amplification initiates in spinous cells in organotypic cultures of primary human keratinocytes during prolonged G(2) phase, as signified by abundant cytoplasmic cyclin B1 (Wang, H. K., Duffy, A. A., Broker, T. R., and Chow, L. T. (2009) Genes Dev. 23, 181-194). In this study, we demonstrated that the E7 protein, which induces S phase reentry in suprabasal cells by destabilizing the p130 pocket protein (Genovese, N. J., Banerjee, N. S., Broker, T. R., and Chow, L. T. (2008) J. Virol. 82, 4862-4873), also elicited extensive G(2) responses. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G(2)/M progression. E7 expression induced cytoplasmic accumulation of cyclin B1 and cdc2 in the suprabasal cells. The elevated cdc2 had inactivating phosphorylation on Thr(14) or Tyr(15), and possibly both, due to an increase in the responsible Wee1 and Myt1 kinases. In cells that harbored cytoplasmic cyclin B1 or cdc2, there was also an accumulation of the phosphatase-inactive cdc25C phosphorylated on Ser(216), unable to activate cdc2. Moreover, E7 expression induced elevated expression of phosphorylated ATM (Ser(1981)) and the downstream phosphorylated Chk1, Chk2, and JNKs, kinases known to inactivate cdc25C. Similar results were observed in primary human keratinocyte raft cultures in which the productive program of HPV-18 took place. Collectively, this study has revealed the mechanisms by which E7 induces prolonged G(2) phase in the differentiated cells following S phase induction.

FIGURE 1.

HPV-18 E7 and genomic HPV-18 plasmid induce elevated expression of cyclin B1 and cdc2 in the differentiated strata of PHK raft cultures. A, double indirect immunofluorescence microscopy was performed to detect cyclin B1 (green) and BrdU (red) in organotypic raft cultures of PHK (left panel), HPV-18 E7 wild type (middle panel), and HPV-18 E7E35Q,E36Q,E37Q (right panel). B, cdc2 (green) and cyclin B1 (red) in raft cultures of HPV-18 E7ΔDLLC (upper row), the wild type HPV-18 E7 (middle row), and HPV-18 genomic plasmid-containing raft cultures (lower row). ΔDLLC and E35Q,E36Q,E37Q are mutations of HPV-18 E7 lacking the pocket protein binding site or CKII motif, respectively. In this figure and Figs. 3 and 5, images were captured at ×20 magnification and arrowheads indicate the basal layer of the epithelia. All the raft cultures shown here are 10 days old.

FIGURE 2.

HPV-18 E7 and genomic HPV-18 plasmid dysregulate cell cycle regulatory proteins favoring a G₂ phase milieu. A, FACS analysis of HeLa cells without any treatment or treated with hydroxyurea (HU) to enrich cells in S phase or with nocodazole (Noc), which enrich cells in M. B–E, Western blot analysis of lysates from asynchronous (AS) HeLa, hydroxyurea-treated HeLa, vector-transduced raft culture (Vector), HPV-18 E7-expressing raft culture (18E7), PHK raft culture containing amplifying HPV-18 genomic plasmids (HPV-18), PHK control raft culture (PHK), and Noc-treated HeLa. B, immunoblots showing steady state levels of HPV-18 E7, cyclin B1, p-cdc2 Thr¹⁴, and p-cdc2 Thr¹⁶¹. C, steady state expression levels of p-cdc2 Tyr¹⁵ and p-cdc2 Thr¹⁶¹. D, steady state expression levels of cdc2 and phospho-histone Ser¹⁰ (p-H3S10). E, steady state expression level of Wee1. F, steady state expression level of unphosphorylated Myt1 (upper panel) and phosphorylated Myt1 (middle panel). B–D and F represent 4 different gels. C and E are from the same gel. The blot represented in panel B was probed, stripped, and reprobed with different antibodies. Actin was used as a protein loading control.

FIGURE 3.

HPV-18 E7 and genomic HPV-18 plasmid induce changes in the expression and phosphorylation status of the cdc25C phosphatase. A, indirect immunofluorescence microscopy was used to detect the expression and localization of p-cdc25C Ser²¹⁶ (red) relative to cdc2 (green) in raft cultures of HPV-18 E7, HPV-18 E7ΔDLLC, HPV-18 E7C27S, HPV-18 E7S32Q,S33Q, and HPV-18 genomic plasmids. B, immunoblot analysis were performed on lysates from vector-only PHK raft cultures, HPV-18 E7-expressing raft cultures, and HPV-18 raft cultures to assess total cdc25C and the phosphorylated cdc25C Ser²¹⁶. C, simultaneous indirect immunofluorescence and HPV-18 DNA fluorescent in situ hybridization to detect HPV-18 DNA amplification (red) relative to p-cdc25C Ser²¹⁶ (green) or cdc2 (green). E7C27S is mutated in the pocket protein binding motif, whereas S32Q,S33Q is mutated in the CKII substrates. Raft cultures in A and B are 10 days old and raft culture in C is 12 days old.

FIGURE 4.

HPV-18 E7 and genomic HPV-18 plasmid activate kinases responsible for cdc25C phosphorylation on Ser²¹⁶. Immunoblot analysis of (A) p-ATR Ser⁴²⁸, p-Chk1 Ser³⁴⁵, total Chk2, and p-Chk2 Thr⁶⁸ and (B) ATM, p-ATM Ser¹⁹⁸¹, total Chk1 from lysates of raft cultures harboring empty vector (Vector), HPV-18 genomic plasmid, or expressing HPV-18 E7 (18E7) or HPV-18 E7ΔDLLC (ΔDLLC) mutation; C, JNK2 and phosphorylated JNKs in raft cultures harboring the vector-only (Vector), HPV-18 E7 (18E7), E7ΔDLLC (ΔDLLC), E7S32Q,E33Q (S32Q,S33Q), or HPV-18 genomic plasmids. HeLa(Noc) denotes lysates from nocodazole-treated HeLa as negative control for JNK2.

FIGURE 5.

A, indirect immunofluorescent detection of p-ATM Ser¹⁹⁸¹ (green) and S phase reentry (BrdU incorporation, red) in vector-only transduced PHK raft culture (top row), HPV-18 E7ΔDLLC mutation-expressing raft culture (second row), HPV-18 E7-expressing raft culture (third row), and HPV-18 plasmid containing raft culture (fourth row). In the last row, the red arrow indicates the high BrdU and low p-ATM Ser¹⁹⁸¹, whereas the green arrow points to high p-ATM Ser¹⁹⁸¹ but low or no BrdU incorporation. B, p-ATM Ser¹⁹⁸¹ expression relative to HPV-18 plasmid amplification was revealed by combined indirect immunofluorescence and HPV-18 DNA fluorescent in situ hybridization. Red arrow indicate high HPV-18 DNA and low or no p-ATM Ser¹⁹⁸¹, whereas the green arrow points to the high p-ATM Ser¹⁹⁸¹ but low or no HPV-18 plasmid. Raft cultures in A are 10 days old and raft culture in B is 12 days old.