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...
Mineralization of the skeleton depends on the balance between levels of pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and phosphate generated from PPi breakdown by alkaline phosphatase (ALP). We report here that ablation of Nf1, encoding the RAS/GTPase–activating protein neurofibromin, in bone–forming cells leads to supraphysiologic PPi accumulation, caused by a chronic ERK–dependent increase in genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank. It also prevents BMP2–induced osteoprogenitor differentiation and, consequently, expression of ALP and PPi breakdown, further contributing to PPi accumulation. The short stature, impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts could be corrected by enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization, suggest that altered PPi homeostasis contributes to the skeletal dysplasiae associated with neurofibromatosis type-1 (NF1), and that some of the NF1 skeletal conditions might be preventable pharmacologically.
The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNAbinding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation.Papillomaviruses are a family of small double-stranded DNA viruses that induce benign and malignant hyperproliferative lesions of the differentiating epithelium (24,25,49). Approximately 25 types of human papillomavirus (HPV) infect the anogenital region. The oncogenic or "high-risk" types, such as HPV16, -18, and -31, are found in the majority of cervical and anal cancers and their precursor lesions (7,25,49). They are also present in a subset of head-and-neck cancers (22, 42). The low-risk types, including HPV6 and -11, cause benign genital warts (condylomas) (29) and laryngeal papillomatosis (6), a rare but debilitating infection acquired by infants during birth from an infected mother.The life cycle of HPV is coupled to the cellular differentiation program that keratinocytes undergo in the epithelium (17,25,32). These viruses infect the basal cell layer, where they maintain their double-stranded DNA genome as a circular episome in the nucleus of infected cells. Maintenance of the HPV episome in primary keratinocyte cultures requires four viral proteins: the two oncogenes, E6 and E7, the E1 replicative helicase, and the multifunctional E2 protein (25, 32). E2 binds to specific sites in the regulatory region of the viral genome to promote its replication, regulate its transcription, and ensure its proper segregation to daughter cells at mitosis (11). E2 is comprised of two functional domains, an N-terminal transactivation domain (TAD) and a C-terminal DNA-binding/dimerization domain separated...
SV40 large T antigen (T-ag)is a multifunctional protein that successively binds to 5-GAGGC-3 sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequencespecific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double-and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5-GAGGC-3 sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ϳ10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.
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