Seiichiro Mori

In Vivo and In Vitro Studies Suggest a Possible Involvement of HPV Infection in the Early Stage of Breast Carcinogenesis via APOBEC3B Induction

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

Biased amplification of human papillomavirus DNA in specimens containing multiple human papillomavirus types by PCR with consensus primers

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)

Inhibition of nuclear entry of HPV16 pseudovirus-packaged DNA by an anti-HPV16 L2 neutralizing antibody.

Rabbit anti-HPV16 L2 serum (anti-P56/75) neutralizes multiple oncogenic human papillomaviruses (HPVs). We inoculated HeLa cells with HPV16 pseudovirus (16PV) and with anti-P56/75-bound 16PV (16PV-Ab). Both 16PV and 16PV-Ab attached equally well to the cell surface. However, the cell-attached L1 protein of 16PV became trypsin-resistant after incubation at 37°C, whereas approximately 20% of the cell-attached 16PV-Ab L1 remained trypsin-sensitive. Confocal microscopy of HeLa cells inoculated with 16PV revealed packaged DNA in the nucleus at 22h after inoculation; however, nuclear DNA was not detected in cells inoculated with 16PV-Ab. Electron microscopy of HeLa cells inoculated with 16PV showed particles located in multivesicular bodies, lamellar bodies, and the cytosol after 4h; no cytosolic particles were detected after inoculation with 16PV-Ab. These data suggest that anti-P56/75 inhibits HPV infection partly by blocking viral entry and primarily by blocking the transport of the viral genome to the nucleus.

Monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein L2.

Antisera induced by immunization of rabbits with the synthetic peptide P56/75, which has the amino acid (aa) sequence from aa56 to aa75 of HPV16 L2, neutralize pseudovirions and raft-virions of multiple high-risk HPV types, indicating that cross-neutralization epitopes are present in the aa56-75 region. We generated two mouse monoclonal antibodies (MAb): MAb13B and MAb24B recognizing the regions of aa64-73 and aa58-64, respectively. The neutralization assay using pseudovirions of HPV16, 18, 31, 33, 35, 51, 52 and 58 showed that MAb13B neutralized HPV16, 18, and 51, and MAb24B neutralized all the types tested. The mixture of MAb13B and MAb24B neutralized HPV16, 18, and 51 pseudovirions more efficiently than each of the MAbs alone. The data indicate that there are at least two cross-neutralization epitopes in the aa56-75 region and that an antigen capable of presenting the two cross-neutralization epitopes would be a good vaccine candidate for a broad-spectrum of HPVs.

Genetic variation of human papillomavirus type 16 in individual clinical specimens revealed by deep sequencing.

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.

Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

BackgroundCo-infection of multiple genotypes of human papillomavirus (HPV) is commonly observed among women with abnormal cervical cytology, but how different HPVs interact with each other in the same cell is not clearly understood. A previous study using cultured keratinocytes revealed that genome replication of one HPV type is inhibited by co-existence of the genome of another HPV type, suggesting that replication interference occurs between different HPV types when co-infected; however, molecular mechanisms underlying inter-type replication interference have not been fully explored.MethodsReplication interference between two most prevalent HPV types, HPV16 and HPV18, was examined in HPV-negative C33A cervical carcinoma cells co-transfected with genomes of HPV16 and HPV18 together with expression plasmids for E1/E2 of both types. Levels of HPV16/18 genome replication were measured by quantitative real-time PCR. Physical interaction between HPV16/18 E1s was assessed by co-immunoprecipitation assays in the cell lysates.ResultsThe replication of HPV16 and HPV18 genomes was suppressed by co-expression of E1/E2 of heterologous types. The interference was mediated by the heterologous E1, but not E2. The oligomerization domain of HPV16 E1 was essential for HPV18 replication inhibition, whereas the helicase domain was dispensable. HPV16 E1 co-precipitated with HPV18 E1 in the cell lysates, and an HPV16 E1 mutant Y379A, which bound to HPV18 E1 less efficiently, failed to inhibit HPV18 replication.ConclusionsCo-infection of a single cell with both HPV16 and HPV18 results in replication interference between them, and physical interaction between the heterologous E1s is responsible for the interference. Heterooligomers composed of HPV16/18 E1s may lack the ability to support HPV genome replication.

Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia

Persistent infection with oncogenic human papillomavirus (HPV) causes cervical cancer. However, viral genetic changes during cervical carcinogenesis are not fully understood. Recent studies have revealed the presence of adenine/thymine‐clustered hypermutation in the long control region of the HPV16 genome in cervical intraepithelial neoplasia (CIN) lesions, and suggested that apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) proteins, which play a key role in innate immunity against retroviral infection, potentially introduce such hypermutation. This study reports for the first time the detection of adenine/thymine‐clustered hypermutation in the E2 gene of HPV16 isolated from clinical specimens with low‐ and high‐grade CIN lesions (CIN1/3). Differential DNA denaturation PCR, which utilizes lower denaturation temperatures to selectively amplify adenine/thymine‐rich DNA, identified clusters of adenine/thymine mutations in the E2 gene in 4 of 11 CIN1 (36.4%), and 6 of 27 CIN3 (22.2%) samples. Interestingly, the number of mutations per sample was higher in CIN3 than in CIN1. Although the relevance of E2 hypermutation in cervical carcinogenesis remains unclear, the observed hypermutation patterns strongly imply involvement of APOBEC3 proteins in editing the HPV16 genome during natural viral infection. J. Med. Virol. 87:1754–1760, 2015.

Identification of APOBEC3B promoter elements responsible for activation by human papillomavirus type 16 E6

Recent cancer genomics studies have identified mutation patterns characteristic of APOBEC3B (A3B) in multiple cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. A3B expression is upregulated by HPV E6/E7 oncoproteins, implying a crucial role for A3B upregulation in HPV-induced carcinogenesis. Here, we explored the molecular mechanisms underlying the activation of the A3B promoter by E6. Luciferase reporter assays with a series of deleted fragments of the human A3B promoter in normal immortalized human keratinocytes (NIKS) identified two functional regions in the promoter: the distal region (from -200 to -51), which is required for basal promoter activity, and the proximal region (from +1 to +45), which exerts an inhibitory effect on gene expression. Each promoter region was found to contain an E6-responsive element(s). Disruption of an AT-rich motif located between +10 and +16 abrogated the proximal-region-mediated activation of the A3B promoter by E6. DNA pull-down assays revealed that a cellular zinc-finger protein, ZNF384, binds to the AT-rich motif in the A3B promoter, and chromatin immunoprecipitation assays confirmed that ZNF384 binds to the A3B promoter in cells. ZNF384 knockdown reduced the A3B mRNA levels in NIKS expressing E6, but not in the parental NIKS, indicating that ZNF384 contributes to A3B upregulation by E6, but not to basal A3B expression. The exogenous expression of ZNF384 led to the activation of the A3B promoter in NIKS. Collectively, these results indicate that E6 activates the A3B promoter through the distal and proximal regions, and that ZNF384 is required for the proximal-region-mediated activation of A3B.

Tissue distribution of cynomolgus adeno-associated viruses AAV10, AAV11, and AAVcy.7 in naturally infected monkeys

SummaryAdeno-associated virus (AAV) is used in gene-therapy studies, but its tissue distribution is unknown in natural infection. We examined cynomolgus AAVs (previously isolated AAV10 and AAV11 and novel AAVcy.7) for their tissue distribution in 14 cynomolgi by type-specific PCR. We found AAV10, AAV11, and AAVcy.7 in 6, 10, and 14 monkeys, respectively, and two or three types in 11 monkeys, showing that these AAVs are widespread in the monkeys. We detected AAV at a higher level mainly in the lymphatic tissues and ileum, which suggests that AAV may invade the host through Peyer’s patches in the ileum and infect immune cells.

Antibody-dependent enhancement of adeno-associated virus infection of human monocytic cell lines.

In host animals, adeno-associated virus (AAV) is detectable mainly in the lymphoid tissue, which appears to be a target in natural infection. We used the human monocytic cell lines THP-1 and U937 to study the effect of mouse anti-AAV2 antiserum on infection with an AAV2 vector having the luciferase gene (AAV2/Luc). AAV2/Luc was found to infect THP-1 and U937 cells much less efficiently than HeLa cells, as monitored with the induced enzyme activity. Pre-incubation of AAV2/Luc with anti-AAV2 antiserum at a sub-neutralizing concentration enhanced by 2-to-10 fold infection of THP-1 and U937 with AAV2/Luc, but not of HeLa. Similarly, anti-AAV10 serum at a low level enhanced infection of THP-1 with AAV10/Luc. Sera of two cynomolgus monkeys, which had been probably infected with an AAV2-like virus, enhanced infection of THP-1 with AAV2/Luc. The enhancement was reduced with blocking the IgG-receptors Fcgamma-RI and Fcgamma-RII, which were displayed on the surface of THP-1 and U937 but not HeLa cells, with anti-Fcgamma-RI antibody or anti-Fcgamma-RII antibody. The data indicate that infection of Fcgamma receptor-bearing cells with AAV is enhanced by anti-AAV IgG antibodies at a sub-neutralizing concentration that play a role in linking AAV particles and Fcgamma receptors.