The results of this study provide pre-vaccination era baseline data on human papillomavirus type distribution in Japanese women and serve as a reliable basis for monitoring the future impact of human papillomavirus vaccination in Japan.
Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair.
Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.
Genotyping human papillomavirus (HPV) in clinical specimens is important because each HPV type has different oncogenic potential. Amplification of HPV DNA by PCR with the consensus primers that are derived from the consensus sequences of the L1 gene has been used widely for the genotyping. As recent studies have shown that the cervical specimens often contain HPV of multiple types, it is necessary to confirm whether the PCR with the consensus primers amplifies multiple types of HPV DNA without bias. We amplified HPV DNA in the test samples by PCR with three commonly used consensus primer pairs (L1C1 ⁄ L1C2+C2M, MY09 ⁄ 11, and GP5+ ⁄ 6+), and the resultant amplicons were identified by hybridization with type-specific probes on a nylon membrane. L1C1 ⁄ L1C2+C2M showed a higher sensitivity than the other primers, as defined by the ability to detect HPV DNA, on test samples containing serially diluted one of HPV16, 18, 51, 52, and 58 plasmids. L1C1 ⁄ L1C2+C2M failed to amplify HPV16 in the mixed test samples containing HPV16, and either 18 or 51. The three consensus primers frequently caused incorrect genotyping in the selected clinical specimens containing HPV16 and one or two of HPV18, 31, 51, 52, and 58. The data indicate that PCR with consensus primers is not suitable for genotyping HPV in specimens containing multiple HPV types, and suggest that the genotyping data obtained by such a method should be carefully interpreted. (Cancer Sci 2011; 102: 1223-1227 H uman papillomavirus (HPV), composed of an icosahedral capsid and a circular double-stranded DNA genome, is classified into more than 100 genotypes based on the nucleotide sequence homology of the L1 gene encoding the major capsid protein.(1) The HPV types found in lesions of the skin and genital mucosa are grouped as cutaneous and genital HPVs, respectively. Of genital HPVs, 15 types (HPV16,18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73) that have been found in cervical cancer are called high-risk HPVs (2) and the types, such as HPV6 and HPV11, that have been found in benign genital warts are called low-risk HPVs. For detection and genotyping of HPV DNA in the clinical specimens, such as cervical swabs and Pap smears, a part of the L1 gene is amplified by PCR then grouped based on the susceptibility to various restriction enzymes, the binding capacity to type-specific probes, or the nucleotide sequences of the amplicons.(4) Several consensus primer pairs have been developed and used as standard primers for PCR-based genotyping of HPV in the clinical specimens. L1C1 ⁄ L1C2+C2M was developed in 1991, (5) and has been used in more than 10 articles describing HPV prevalence in the Japanese population.(5-16) MY09 ⁄ 11 (17) and GP5+ ⁄ 6+ (18) were developed in 1989 and 1995, respectively, and have been used in numerous studies worldwide. (19) These primers are derived from the consensus sequences of the L1 gene and the amplicons contain type-specific sequences. Recently new primers, PGMY09 ⁄ 11 (20) and modified GP5+ ⁄ 6+ (MGP), (21) which are composed...
Replication of human papillomavirus (HPV) genomes requires an origin of replication and two viral proteins: the DNA helicase E1 and the auxiliary factor E2. To dissect the profile of HPV replication in the epithelium, we analyzed replication of an HPV16 origin-containing plasmid in human epithelial cell extracts supplemented with purified E1 and E2. We found that in addition to well-defined circular replication products, high-molecular-weight DNA was synthesized in a manner that depended on the origin, E1 and E2. The high-molecular-weight DNA was converted to a unit-length linear DNA by treatment with restriction enzymes that cleave the plasmid once, implying that a concatemeric DNA was generated by rolling circle replication. Nicking or relaxing the template plasmid enhanced the level of HPV rolling circle replication. In contrast, the addition of an extract from non-epithelial cells diminished the generation of the rolling circle replication product in the epithelial cell extract, indicating factors that counteract HPV rolling circle replication. These results suggest a rolling circle replication mechanism for the HPV genome in cervical epithelial cells, which may have physiological implications for generation of the tandem-repeated HPV genomes occasionally found integrated into the chromosome of cervical cancer cells. IntroductionHuman papillomaviruses (HPVs) are small icosahedral viruses that contain a double-stranded circular DNA genome of approximately 8000 bp (zur Hausen 1996). Among more than 100 types of HPVs so far identified, nearly 15 types are recognized as high-risk types that are closely linked to the development of cervical cancer, with HPV type 16 (HPV16) the predominant high-risk type worldwide (Parkin et al. 2008). HPV infects the basal cells of the epithelium and its genome is maintained as episomal plasmids in the nucleus such that HPV establishes latent infection. When the infected cells leave the basal layer and commence terminal differentiation of the epithelium, the viral genome starts to replicate to yield many thousands of progeny viruses. Thus, replication of the HPV genome is regulated in a strict way that depends on the differentiation status of the host epithelial cells (Longworth & Laimins 2004).The replication of the HPV genome requires an origin of replication and two virally encoded proteins: the DNA helicase E1 and the replication ⁄ transcription factor E2 (Kadaja et al. 2009). To initiate viral DNA replication, E2 binds to a specific binding site at the origin and then recruits E1, leading to the assembly of double E1 hexamers. The resultant E1 hexamer is an active replicative helicase that can induce melting of the DNA at the origin as well as subsequent unwinding of the double helical DNA during replication fork progression. With the exception of continuous DNA unwinding by E1, HPV uses host replication proteins, such as DNA polymerases, proliferating cell nuclear antigen and replication protein A, to accomplish its genome replication (Park et al. 1994;Melendy et al. 1995)....
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