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. 1999 Jan 19;96(2):382-7.
doi: 10.1073/pnas.96.2.382.

Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication

T Ma  1 , N Zou, B Y Lin, L T Chow, J W Harper
Affiliations

Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication

T Ma et al. Proc Natl Acad Sci U S A. .

Abstract

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.

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Figures

Figure 1
Figure 1
Cyclin E interacts with HPV E1. (a) Cdk-independent interaction of E1 and E1/E2 complexes with cyclin E in insect cells. Cells were infected with the indicated viruses and complexes were analyzed by SDS/PAGE and Coomassie staining after purification using GSH–Sepharose (lanes 1–10). Proteins were confirmed by immunoblotting (data not shown). In lanes 11–14, lysates from cells expressing E1, E2, or E1/E2 were mixed with cyclin E-containing lysates before purification. (b) Association of cyclin E/Cdk2 and E1 in COS-1 cells. Lysates from cells transfected with the indicated plasmids (Right) were subjected to immunoprecipitation and immunoblotting by using the indicated antibodies (Left and Center). Quantities of plasmids were: pMT2-E1*, 10 μg; pCMV-cyclin E, 1 μg; pCMV-Cdk2, 1 μg plus empty pMT2 to maintain DNA levels.
Figure 2
Figure 2
Multiple cyclins associate with and phosphorylate E1 and E2 in vitro. (a) Purified cyclin/Cdk complexes or GST–p21 as a negative control were immobilized on GSH–Sepharose beads and used in binding reactions with lysates containing EE–E1/E2 complexes or uninfected lysates as indicated. Complexes were immunoblotted with antibodies against EE–E1, E2, or GST (lanes 1–14). An anti-E1 immune complex (lanes 15 and 16) served to control for the EE–E1/E2 interaction. (b) EE–E1/E2/cyclin complexes prepared as described in a were used in kinase assays. For assays containing kinase and E1, mixtures were fractionated into EE–E1 bound complexes (lanes 2, 6, 10, 14, 18, and 20) and reaction supernatants (∗; lanes 3, 7, 11, 15, 19, and 21) before SDS/PAGE and immunoblotting. Kinase activity was visualized by autoradiography of the filter and proteins identified by immunoblotting with anti-E1 and -E2 antibodies. The positions of phosphorylated EE–E1(>) and E2 (<) are indicated.
Figure 3
Figure 3
An RXL motif in E1 is required for association with and phosphorylation by cyclin E/Cdk2. (a) Domains and positions of candidate Cdk phosphorylation sites in HPV E1 are shown. (b) Mutations in the RXL motif of E1 abolish association with cyclin E in vitro. Equal quantities (0.8 μg) of EE–E1 (lanes 1–5), EE–E1ARA (lanes 6–10), and EE–E1ΔCdk (lanes 11–15) were used for binding with increasing quantities of insect-cell lysates with or without cyclin E/Cdk2 and bound proteins detected by immunoblotting. To control for levels of input proteins, immunoblots of reaction mixtures prior to bead washing were performed (Top). (c) EE–E1 (lanes 1–4), EE–E1ARA (lanes 5–8), and EE–E1ΔCdk (lanes 9–12) immobilized on anti-EE beads were incubated with purified cyclin E/Cdk2 (1, 10, and 100 nM, respectively) and [γ-32P]ATP (see Materials and Methods). Products were separated by using SDS/PAGE and transferred to nitrocellulose before immunoblotting (Top) and autoradiography (Bottom). (d) E1ARA and E1ΔCdk interact with E2. Anti-EE immune complexes of insect-cell lysates derived from the indicated infections were separated by using SDS/PAGE and visualized with Coomassie blue. (e) Immobilized EE–E1 or EE–E1/E2 complexes were assembled with cyclin E/Cdk2 in vitro and the excess kinase removed before assay in the presence (lanes 5–8) or absence (lanes 1–4) of histone H1. As a control, serial dilutions of cyclin E/Cdk2 (1–1,000 ng) were used in histone H1 kinase assays (lanes 9–12).
Figure 4
Figure 4
Efficient HPV replication requires an intact cyclin-binding motif and Cdk-phosphorylation sites in E1. (a and b) HPV replication assays were performed as described (18, 20). Low molecular weight DNA was analyzed by Southern blotting using labeled origin plasmid as a probe to detect the presence of DpnI-resistant replication products. This probe contains sequences that also hybridize with E1 and E2 plasmids. Blots were quantified by PhosphorImager analysis. E1* reflects the use of pMT2-E1* based plasmids, which express higher levels of protein and replication activity. (c) Levels of E1*, E1*ARA, and E1*ΔCdk proteins were determined by immunoblotting with anti-E1 antibodies. (d) Cell-free replication was conducted as described in Material and Methods and products detected by incorporation of [α-32P]dCTP.
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