Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses

Abstract

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...

Biology of Human Papillomavirus Infections in Head and Neck Carcinogenesis

“…The HPV types have also been grouped into mucosal or cutaneous types, based on their tropism for specific epithelial sites. Mucosal HPV types found preferentially in precancerous and cancerous lesions have been designated as 'high-risk' types and these include types 16,18,31,33,34,35,39,45,51,52,56,58,59, 66, 68, and 70. Mucosal HPVs found in benign genital warts and other non-malignant lesions are generally labeled as 'low-risk' types, the most important ones being HPV 6,11,42,43, and 44 [1,2].…”

“…The HPV E7 gene can immortalize human keratinocytes alone when expressed at high levels [for review see 24,34,35]. E7 protein of the low-risk HPV types also binds to pRb, although with lower affinity, but thus indicating its significance in transformation.…”

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy

“…Different from other N-recognins, UBR4 does not have a known ubiquitylation domain (7), suggesting that the action mechanism of UBR4 in N-end rule degradation could be different from those of canonical N-recognins. Recent studies using cultured cells suggest that UBR4 plays a role in membrane morphogenesis and extracellular matrix-mediated cell survival and signaling (15), human papillomavirus (HPV) E7-mediated cellular immortalization (16)(17)(18), and migration and development of neurons (19).…”

Identification and Proteomic Analysis of Distinct UBE3A/E6AP Protein Complexes

“…These MS data were then analyzed with CompPASS (Comparative Proteomic Analysis Software Suite), which relies on an unbiased comparative approach for identifying high-confidence candidate-interacting proteins (HCIPs) (85). This technology has been successfully used to identify protein interactors among the hundreds of proteins typically identified in immunoprecipitation-MS experiments (5,12,72,73,95).…”