Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

Marcel Dreer,Johannes Madlung,Elke Straub,Frank Stubenrauch,Jasmin Fertey,Boris Macek,Saskia Van De Poel,Thomas Iftner,Paul Francis Lambert

doi:10.1371/journal.ppat.1005556

Abstract

Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.

Highlights

Human papillomaviruses (HPV) constitute one of the largest human pathogenic virus families known to date
Previous studies have suggested that some HPV encode an E8^E2C protein that limits genome replication in undifferentiated cells
We demonstrate that E8^E2C proteins from phylogenetically diverse HPV types interact with NCoR/SMRT corepressor complexes to limit viral transcription and genome replication

Summary

Introduction

Human papillomaviruses (HPV) constitute one of the largest human pathogenic virus families known to date. Viral proteins derived from the E1 and E2 genes function as sequence-specific DNA binding proteins and are involved in the initiation of DNA replication, control of viral transcription and segregation of viral genomes [5,6,7]. E2 can act either as a transcriptional repressor or activator which is dependent on the location of E2 binding sites (E2BS) in E2-responsive promoters and may control replication by modulating viral gene expression [6]. The amino terminal domain of E2 (~200 residues) is required for the activation of replication, modulation of transcription and attachment of PV genomes to mitotic chromosomes [6] These functions are mediated by interaction with viral E1 and cellular proteins such as Brd which contact E2 via highly conserved residues among PV [8,9,10,11]

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