Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts.

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.

Do neutralizing antibody responses generated by human papillomavirus infections favor a better outcome of low-grade cervical lesions?

To determine the role of neutralizing antibody generated by human papillomavirus (HPV) infections, baseline levels of serum neutralizing antibodies directed against HPV 16 and cervical HPV DNA were determined in 242 unvaccinated women with low-grade cervical abnormalities, who were then monitored by cytology and colposcopy every 4 months. In women infected with HPV 16 (n = 42), abnormal cytology persisted longer in those positive for HPV 16-specific neutralizing antibodies at baseline (median time to cytological regression: 23.8 vs. 7.2 months). Progression to cervical precancer (cervical intraepithelial neoplasia grade 3) within 5 years occurred only among women carrying HPV 16-specific neutralizing antibodies (P = 0.03, log-rank test). In women infected with types other than HPV 16 (n = 200), detection of HPV 16-specific neutralizing antibodies was not correlated with disease outcome. In conclusion, development of specific neutralizing antibodies following natural HPV 16 infection did not favor a better outcome of low-grade cervical lesions induced by HPV 16 or by other types; rather, detection of neutralizing antibodies generated by current infection may reflect viral persistence and thus help identify those who are at high risk of disease progression.

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.

Divergent evolution of norovirus GII/4 by genome recombination from May 2006 to February 2009 in Japan.

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P

Enhanced transgene expression in the mouse skeletal muscle infected by the adeno-associated viral vector with the human elongation factor 1alpha promoter and a human chromatin insulator.

BACKGROUND: Efficient and continuous expression of a therapeutic transgene is a key factor for improving the efficacy of gene therapy. Some insulators are known to contribute to continuous high-level expression of a therapeutic transgene. METHODS: Using the human AAVS1 insulator (DHS) found in the AAVS1 DNase I hypersensitive site, chicken beta-globin insulator (cHS4) and sea urchin arylsufatase insulator (Ars), we newly constructed three recombinant adeno-associated virus vectors (rAAV) and examined their capability of transducing the mouse quadriceps muscle. RESULTS: DHS increased transgene expression from the human elongation factor 1alpha promoter (EF) by 1000-fold, up to the high level achieved by the human cytomegalovirus immediate early promoter/enhancer (CMV), which comprises an extremely strong promoter for driving a transgene. cHS4 enhanced the expression by 100-fold, whereas Ars did not. The enhanced expression was maintained for at least 24 weeks. Vector copy numbers were similar with and without DHS or cHS4; thus, the enhancement is most likely due to up-regulated transcription. Neither DHS, nor cHS4 affected transgene expression from CMV. DHS enhanced expression from the human muscle creatine kinase promoter/enhancer by 100-fold in mice, as did DHS from EF. CONCLUSIONS: Although DHS was unable to further enhance high expression from the strong viral enhancer/promoter, it enhanced low expression from the human promoters by 100- to 1000-fold. Thus, DHS may be useful for constructing rAAVs that express a therapeutic transgene from less efficient, tissue specific promoters.

Identification of nucleolin as a protein that binds to human papillomavirus type 16 DNA.

Transcription, replication, and segregation of human papillomaviruses (HPVs) are regulated by various host factors, but our understanding of host proteins that bind to the HPV genome is limited. Here we report the results of a search of cellular proteins that can associate with specific genomic regions of HPV type 16 (HPV16). We found that human nucleolin, an abundant nucleolar protein, was preferentially captured in vitro by an HPV16 genomic fragment from nucleotide positions (nt) 531-780. Electrophoretic mobility shift assays with a bacterially expressed nucleolin revealed that nucleolin bound to an HPV16 genomic region between nt 604 and 614 in a sequence-dependent manner. Chromatin immunoprecipitation analysis showed that both exogenous and endogenous nucleolin bound to a plasmid containing the HPV16 genomic region in HeLa cells. Furthermore, nucleolin associated with the HPV16 genome stably maintained in HPV16-infected W12 cells, suggesting that the nucleolin binding may be involved in the dynamics of the HPV genome in cells.

Identification of monomorphic and divergent haplotypes in the 2006-2007 norovirus GII/4 epidemic population by genomewide tracing of evolutionary history.

Our norovirus (NoV) surveillance group reported a >4-fold increase in NoV infection in Japan during the winter of 2006-2007 compared to the previous winter. Because the increase was not linked to changes in the surveillance system, we suspected the emergence of new NoV GII/4 epidemic variants. To obtain information on viral changes, we conducted full-length genomic analysis. Stool specimens from 55 acute gastroenteritis patients of various ages were collected at 11 sites in Japan between May 2006 and January 2007. Direct sequencing of long PCR products revealed 37 GII/4 genome sequences. Phylogenetic study of viral genome and partial sequences showed that the two new GII/4 variants in Europe, termed 2006a and 2006b, initially coexisted as minorities in early 2006 in Japan and that 2006b alone had dominated over the resident GII/4 variants during 2006. A combination of phylogenetic and entropy analyses revealed for the first time the unique amino acid substitutions in all eight proteins of the new epidemic strains. These data and computer-assisted structural study of the NoV capsid protein are compatible with a model of antigenic drift with tuning of the structure and functions of multiple proteins for the global outgrowth of new GII/4 variants. The availability of comprehensive information on genome sequences and unique protein changes of the recent global epidemic variants will allow studies of diagnostic assays, molecular epidemiology, molecular biology, and adaptive changes of NoV in nature.

Development of an HPV vaccine for a broad spectrum of high-risk types.

Cervical cancer, the second most frequent gynecological malignancy in the world, is caused by infection with high-risk human papillomaviruses (HPV16, 18, and other 13 types). Vaccines protecting women from infection with high-risk HPVs can reduce prevalence of cervical cancer without costly screening for cervical precancerous lesions. Two vaccines containing HPV16 and 18 virus-like particles (VLPs), which were produced by self-assembly of the major capsid protein L1 of each HPV type, successfully induced type-specific neutralizing antibodies in the recipients of the large scale clinical trials and have been approved by several countries. Although HPV16 and HPV18 account for approximately 50% and 20% of cervical cancer, respectively, the remaining major issue to be addressed is how to prevent infection with other high-risk HPVs. Our and other studies have indicated that the HPV minor capsid protein L2 has several type-common neutralization epitopes and that immunization of animals with peptides having the L2-epitopes protects them against experimental challenge. Recently, we have shown that a type of chimeric VLP, HPV16 VLPs to which the type-common L2-epitope was added, induced in rabbits both the anti-HPV16 L1 neutralizing antibody at a high level and the anti-L2 cross-neutralizing antibody at lower but sufficient levels to be protective shown in the other animal experiments. Thus, this type of chimeric VLP seems likely to be one of the next-generation vaccine candidates for a broad spectrum of high-risk HPV types. Future issues were discussed about the HPV vaccine development.

Transcription factor human Skn-1a enhances replication of human papillomavirus DNA through the direct binding to two sites near the viral replication origin.

Human papillomavirus type 16 (HPV16) DNA replication, which requires two viral proteins E1 and E2, occurs only in the differentiating epithelium. Besides the general factors necessary for cellular DNA synthesis, other unidentified cellular factors are assumed to be involved in the regulation of HPV DNA replication. In the present study, we found that the POU-domain transcription factor human Skn-1a, which induces the terminal differentiation of keratinocytes and activates the HPV16 late promoter, enhanced the transient replication of a plasmid containing the HPV16 replication origin in HEK293 cells when co-transfected with a plasmid expressing E1 and E2. An electrophoretic mobility shift assay with a bacterially expressed human Skn-1a or an extract of HeLa cells over-expressing human Skn-1a revealed the presence of two human Skn-1a binding sites that are distinct from the known three sites, near the replication origin. A chromatin immunoprecipitation analysis showed that human Skn-1a bound to these sites in cells. Nucleotide substitutions in the sites abolished the binding of human Skn-1a and the human Skn-1a-mediated replication enhancement. The data strongly suggest that, through the binding to the two sites, human Skn-1a enhances HPV DNA replication.

Characterization of mouse 3T3-swiss albino cells available in Japan: necessity of quality control when used as feeders.

Mouse 3T3-Swiss albino cells are widely used as feeder cells to culture the human epidermis for the treatment of burns. To minimize the risk of xenoinfection, quality control of the feeder cells is required in the Japanese guidelines on regenerative medicine using feeder cells. We characterized three lots of 3T3-Swiss albino cells that are publicly or commercially available in Japan. One lot, which propagated more rapidly than the other two without showing typical contact inhibition, was found to release endogenous murine leukemia virus upon iododeoxyuridine-treatment. Southern blotting of restriction fragments showed that the rapidly growing lot consisted of genetically altered cells that had probably emerged during the passages. The data support the guidelines that recommend the quality control of each lot of 3T3-Swiss albino cells if they are to be used clinically.

Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1.

BACKGROUND: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. RESULTS: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. CONCLUSION: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

IgG antibodies to HPV16, 52, 58 and 6 L1-capsids and spontaneous regression of cervical intraepithelial neoplasia.

To identify the predictive markers for spontaneous regression of cervical intraepithelial neoplasia (CIN), we examined whether IgG antibody responses to common human papillomavirus (HPV) L1-capsids correlate with CIN regression. In a cohort study, a total of 116 Japanese women with CIN grade I/II were tested for cervical HPV DNA and serum IgG antibodies to HPV16/52/58/6 L1-capsids. Our data suggest that baseline IgG reactivities to HPV L1-capsids do not serve as a predictive marker of CIN regression, in contrast to histological CIN grades and HPV DNA status.

Biodistribution of a low dose of intravenously administered AAV-2, 10, and 11 vectors to cynomolgus monkeys.

In gene therapy trials, adeno-associated virus (AAV) vectors are injected directly into target tissues such as muscle and liver. Direct injection can lead to the introduction of a low level of the vector into blood circulation. To determine the systemic effects of the vector released in the blood, we extensively examined the biodistribution of intravenously administered AAV serotype 2 (AAV2) vector in cynomolgus monkeys. Although the vector distribution pattern varied from monkey to monkey, the vector DNA was maintained in the various tissues beyond 7 months post-inoculation (pi). The vector DNA was detected in the lymphoid tissues, particularly in the spleen, more frequently and at a much higher level than in the other tissues tested (i.e., brain, lung, liver, heart, gallbladder, pancreas, colon, kidney, ovary, uterus, etc.). The expression of a transgene was detected in the lymph nodes at 3 months pi. The distribution of two pseudotyped vectors, AAV2/10 and AAV2/11, was similar to that of the AAV2 vector. The present results suggest that when introduced intravenously, the AAV vector DNA persists and may induce transgene expression in various monkey tissues. Thus, the possibility of inadvertent gene transfer to various non-target tissues should be considered in a gene therapy strategy with an AAV vector.

[Human papillomavirus and cervical cancer].

Human papillomavirus (HPV) is a small non-enveloped icosahedral virus with a circular double-stranded DNA genome of 8 kilo base pairs. HPV particles reach and infect the basal cells of the stratified epithelia through small epithelial lesions. In the basal cells the viral DNA is maintained as episomes, which start to replicate when the host cells initiate terminal differentiation. In these differentiating cells the degradation of p53 by the E6 protein and the abrogation of the pRb functions by the E7 protein lead to the reactivation of the DNA synthesis machinery. After virus propagation the host cells usually die. On the other hand, in some of the infected cells, the E6 and E7 genes are integrated on rare occasion into cell DNA. The cell continuously expressing the E6 and E7 proteins from the integrated genes is immortalized and sometimes acquires malignant phenotype induced by the accumulated damages to DNA. Of more than 100 HPV genotypes recorded to date, 13 including types 16 and 18 are associated with cervical cancer. Expression of HPV major capsid protein L1 in some cultured cells results in production of virus-like particles (VLPs). The VLPs of types 6, 11, 16, and 18 were used as a prophylactic vaccine in recent clinical trials and shown to successfully induce type-specific neutralizing antibodies in the recipients.

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells.

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness.

Keratinocyte-specific POU transcription factor hSkn-1a represses the growth of cervical cancer cell lines.

The POU transcription factor human Skn-1a (hSkn-1a) specifically promotes the proliferation of keratinocytes and enhances their differentiation. We examined the effects of hSkn-1a on cervical cancer cell lines of epithelial origin, in which the differentiation program is interrupted. From HeLa/Tet-On, a clone that can be induced to make hSkn-1a by doxycycline (HeLa/hSkn-1a) was prepared and characterized. Shortly after the induction, the cells expressed cytokeratin 10 (K10), a major marker protein in differentiating keratinocytes. While maintained for several days in the presence of doxycycline, the HeLa/hSkn-1a cells showed a slightly prolonged time of population doubling, the occasional appearance of flat cells with lowered DNA synthesis, and a low level of apoptotic DNA fragmentation. In SiHa and HeLa S3 cultures, K10 mRNA and apoptotic DNA fragmentation were detected at 48 h after infection with an adenoviral vector capable of expressing hSkn-1a. A colony inhibition assay showed that the growth of HeLa S3, SiHa, CaSki, and C-33A cells was repressed, as seen from the decreased number and average size of the drug-resistant colonies at 2 or 3 weeks after transfection with a plasmid that can express hSkn-1a and neomycin resistance gene. These results suggest that the expression of hSkn-1a represses the growth of the cervical cancer cells through the partial resumption of the differentiation pathway followed by slow suppression of cell replication and apoptosis.

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.

Structural basis for specific recognition of substrates by sapovirus protease.

Sapovirus (SaV) protease catalyzes cleavage of the peptide bonds at six sites of a viral polyprotein for the viral replication and maturation. However, the mechanisms by which the protease recognizes the distinct sequences of the six cleavage sites remain poorly understood. Here we examined this issue by computational and experimental approaches. A structural modeling and docking study disclosed two small clefts on the SaV protease cavity that allow the stable and functional binding of substrates to the catalytic cavity via aromatic stacking and electrostatic interactions. An information entropy study and a site-directed mutagenesis study consistently suggested variability of the two clefts under functional constraints. Using this information, we identified three chemical compounds that had structural and spatial features resembling those of the substrate amino acid residues bound to the two clefts and that exhibited an inhibitory effect on SaV protease in vitro. These results suggest that the two clefts provide structural base points to realize the functional binding of various substrates.

Genotype distribution of human papillomaviruses in Japanese women with abnormal cervical cytology.

We report the prevalence and genotype distribution of human papillomaviruses (HPVs) among Japanese women with abnormal cervical cytology using the PGMY-CHUV assay, one of PGMY-PCR-based lineblot assays that was validated and shown to be suitable for the detection of multiple HPV types in a specimen with minimum bias. Total DNA was extracted from cervical exfoliated cells collected from 326 outpatients with abnormal Pap smears. Overall, 307 specimens (94%) were HPV-positive, 30% of which contained multiple genotypes. The prevalence of HPV DNA was 83% (49/59 samples) in atypical squamous cells of undetermined significance (ASC-US); 91% (20/22 samples) in atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); 97% (130/134 samples) in low-grade squamous intraepithelial lesion (LSIL); and 99% (85/86 samples) in high-grade squamous intraepithelial lesion (HSIL). Three most frequent HPV types detected in HSIL were HPV16 (36%), HPV52 (24%), and HPV58 (14%). Our results suggest that multiple HPV infections are more prevalent in Japanese women than previously reported, and confirm that HPV52 and 58 are more dominant in their cervical precancerous lesions when compared to those reported in Western countries.