We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.T he human papillomaviruses (HPVs) comprise more than 120 different virus types, each with a double-stranded DNA genome of approximately 8 kb. The HPVs have similar genome organizations, with regulatory functions encoded by the early (E) genes and structural components encoded by the late (L) genes (reviewed in reference 24). HPVs of different types differ in DNA sequences by 10% or more in the L1 gene, and the other viral genes exhibit a greater degree of sequence diversity between types (6, 15). Papillomaviruses are grouped into genera based on their L1 gene sequences and are further subdivided into species, with most of the HPVs in genus alpha or genus beta. The genus alpha HPVs infect the mucosal epithelium, and those that are the etiological agent responsible for the development of anogenital cancers (including cervical cancer) fall into genus alpha, species 7, and genus alpha, species 9. Beta-type HPVs infect the cutaneous epithelium.The HPV E6 protein has long been appreciated as a critical regulator of the viral life cycle and driver of tumorigenesis for the high-risk HPVs. Through the action of the HPV E7 protein, the G 1 -S checkpoint is bypassed in infected cells by the inactivation of the retinoblastoma tumor suppressor protein (pRB1) (17,18,46). This results in a cellular environment that is conducive to the replication of the viral DNA but in which proapoptotic signals have been triggered. One crucial function of high-risk HPV E6 proteins is to counteract the effects of p53 following this trigger, and this is accomplished by the targeted ubiquitylati...

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.T he many types of human papillomaviruses (HPVs) that have been described exhibit considerable diversity. The HPVs are DNA viruses with a tropism specific for squamous epithelial cells. More than 120 HPVs have been identified and cloned to date, and these share a conserved genomic structure with eight to 10 ORFs encoded on one strand of a small double-stranded circular DNA genome (1). The ORFs involved in fundamental processes such as DNA replication or capsid formation are well conserved. Other ORFs, such as E6 and E7, have some conserved features but are more divergent at the nucleotide and protein level. Consistent with these differences, the lesions that are caused by infection with different HPVs and the propensity for these lesions to progress to cancer vary as well (2). A subset of the HPVs are the primary etiological agent in the development of cervical cancer, and other HPVs cause genital or cutaneous warts or other skin lesions. Relatively little is known about how these sequence differences translate into different biological outcomes in infected human cells. Thus, there exists an opportunity to systematically define features of diverse HPVs and to understand at the molecular level how their varied genetic compositions result in different disease states.The standard phylogeny of the HPVs is based on the sequence of the L1 gene, and a virus with an L1 DNA sequence that differs by 10% or more from other HPV L1s is designated as a separate type (1). Similar HPV types are grouped into species and further into genera. The majority of the HPVs identi...

The Notch signaling pathway is a key determinant in keratinocyte differentiation and growth cycle arrest, and has been reported to have a tumor suppressor function in skin. The papillomavirus life cycle is intricately linked to the differentiation status of keratinocytes. Papillomaviruses are associated with benign proliferative epithelial lesions in their respective hosts. Although human papillomaviruses (HPVs) associated with genital tract lesions have been extensively studied, studies of the cutaneous HPVs are more limited. In particular, it is well established that the E6 proteins of high-risk HPVs of the α-genus such as HPV16 and HPV18 mediate the degradation of p53 by its association with the ubiquitin ligase E6AP. In contrast, less is known about the cellular activities of the cutaneous HPVs of the β-genus. By using an unbiased proteomic approach, we identify MAML1 and other members of the Notch transcription complex as high-confidence cellular interacting proteins of E6 proteins of the β-genus HPVs and of the bovine papillomavirus type 1 associated with cutaneous fibropapillomas. We show that bovine papillomavirus type 1 and β-HPV E6 repress Notch transcriptional activation, and that this repression is dependent on an interaction with MAML1. Finally, we show that the expression levels of endogenous Notch target genes are repressed by β-HPV E6 proteins. These findings elucidate a mechanism of viral antagonism of Notch signaling, and suggest that Notch signaling is an important epithelial cell pathway target for the β-HPVs.

The capability to directly interrogate intracellular structures inside a single cell for measurement and manipulation is important for understanding subcellular and suborganelle activities, diagnosing diseases, and developing new therapeutic approaches. Compared with measurements of single cells, physical measurement and manipulation of subcellular structures and organelles remain underexplored. To improve intracellular physical measurement and manipulation, we have developed a multipole magnetic tweezers system for micromanipulation involving submicrometer position control and piconewton force control of a submicrometer magnetic bead inside a single cell for measurement in different locations (spatial) and different time points (temporal). The bead was three-dimensionally positioned in the cell using a generalized predictive controller that addresses the control challenge caused by the low bandwidth of visual feedback from high-resolution confocal imaging. The average positioning error was quantified to be 0.4 μm, slightly larger than the Brownian motion–imposed constraint (0.31 μm). The system is also capable of applying a force up to 60 pN with a resolution of 4 pN for a period of time longer than 30 min. The measurement results revealed that significantly higher stiffness exists in the nucleus’ major axis than in the minor axis. This stiffness polarity is likely attributed to the aligned actin filament. We also showed that the nucleus stiffens upon the application of an intracellularly applied force, which can be attributed to the response of structural protein lamin A/C and the intracellular stress fiber actin filaments.