Human Papillomavirus E1 Helicase Interacts with the WD Repeat Protein p80 To Promote Maintenance of the Viral Genome in Keratinocytes

Abstract

Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.Papillomaviruses (PV) are small, nonenveloped, doublestranded DNA viruses that induce hyperproliferative lesions of cutaneous and mucosal stratified squamous epithelia. They can infect the majority of higher vertebrates, with humans being the host for more than 100 different PV types. Based on oncogenic propensity, human PV (HPV) types that infect the anogenital region are categorized as either low-risk types, such as HPV type 6 (HPV6) and HPV11, which cause genital warts, or high-risk types, like HPV16, -18, and -31, which induce dysplasic lesions that can progress to cancer. The viral replication cycle is tightly linked to the differentiation program of stratified epithelia, the expression of viral proteins being modulated by intracellular changes occurring as cells differentiate toward the upper layers of the epithelium. The viral replication cycle begins with the infection of an undifferentiated basal cell in which the viral genome is established as an extrachromosomal episome in the nucleus at 50 to 100 copies, a number that is maintained until infected keratinocytes reach the upper cell layers (reviewed in reference 15). In those differentiated cells, the viral genome is amplified to a high copy number, probably as a result of increased expression of the viral replicative helicase E1, brought about by differentiation-dependent activation of the late promoter (20, 34). The viral structural proteins, which are also expressed from the late promoter, are produced in the upper lay...

Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

“…The plasmid used to express untagged E1 was constructed by excising the 3ϫ Flag coding region from pCMV-3Tag-1a by NotI/BamHI digestion and replacing it with the codon-optimized E1 ORF. The plasmids expressing the red fluorescent protein (RFP) and RFP-p80 have been described previously (12). The RFP-E2 expression plasmid was constructed by PCR amplification of the codon-optimized E2 ORF and ligation between the NotI and XbaI sites of the RFP vector.…”

“…Rabbit polyclonal antibodies against p80 or E1 were raised, respectively, by injecting rabbits (Open Biosystems) with a purified C-terminal fragment of p80 (amino acids 400 to 677) or an E1 fragment (amino acids 50 to 332) produced in bacteria as a GST-6ϫHis fusion protein. Protein purification and cleavage of the GST moiety were performed as previously described (12). For Western blot analysis, proteins were transferred onto polyvinylidene difluoride membranes and were detected using horseradish peroxidase-conjugated secondary antibodies from GE Healthcare, either sheep anti-mouse IgG (catalog no.…”

“…1A) that prevent p80 binding were identified. These substitutions were found to abrogate the maintenance of the viral genome in immor-talized keratinocytes and to reduce transient HPV DNA replication (12,20). The substitutions appeared to specifically prevent the interaction of E1 with p80, as they affected neither the ability of E1 to bind to DNA or to Cdk2 in vitro nor its nuclear accumulation in vivo in transfected cells.…”

“…Altogether, these sequences mediate the nucleocytoplasmic shuttling of E1 and its nuclear accumulation during S phase, when viral DNA replication occurs (14,19). We previously reported that the N-terminal regulatory region of E1 from anogenital HPV types also interacts with the cellular protein p80 (12), alternatively known as UAF1 or WDR48. Deletion analysis revealed that amino acids (aa) 10 to 40 of E1, a region that is well conserved among low-and high-risk HPV genotypes, constitutes the minimal p80-binding domain (Fig.…”

Mapping the Disease Protein Interactome: Toward a Molecular Medicine GPS to Accelerate Drug and Biomarker Discovery

“…For this purpose, we like combining the use of HEK293 cells, for which we and others have already generated significant, although far from complete, protein-protein interaction data, with cell lines specifically originating from the disease/tissue of interest. Mapping interaction networks generally identifies both upstream (i.e., regulators) and downstream (i.e., regulated proteins) effectors of the tagged proteins, as we have shown for components of the transcription machinery [3][4][5]8,15,16,18 and as others have shown for proteins specifically involved in disease conditions. 2,6,10,12,27,32,33 Consequently, given that a protein associated with a specific disease condition represents a putative biomarker for this particular disease, its interactors putatively represent additional biomarkers that can regulate the disease phenotype by acting as upstream or downstream effectors.…”