DNA Replication of Human Papillomavirus Type 31 Is Modulated by Elements of the Upstream Regulatory Region That Lie 5′ of the Minimal Origin

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5′-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5′-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5′-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.     

Induction of the upstream regulatory region of human papillomavirus type 31 by dexamethasone is differentiation dependent

Glucocorticoids have been shown to play a role in the transforming abilities of human papillomaviruses (HPVs), and glucocorticoid response elements (GREs) have been identified in the upstream regulatory regions (URRs) of various HPV types. These findings have made glucocorticoids potential therapeutic targets for HPV infection. We have previously shown that the URR of HPV type 31 (HPV31) is insensitive to induction by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture, despite the identification of three potential GREs in the 5' region of the URR. Due to the fact that the HPV life cycle is intimately linked to the differentiation of the host tissue, we chose to determine whether the URR of HPV31 was inducible by dex under differentiating conditions. Upon suspension of cells in a semisolid medium of methylcellulose, we found that the URR of HPV31 was inducible by dex. The three GREs appear to play roles as independent repressors of this inducibility. By 5' deletion analysis, the element(s) responsible for this induction was localized to nucleotides (nt) 7238 to 7557. Furthermore, we found that the region between nt 7883 and 7900 appears to act as a repressor of dex inducibility. These findings indicate that epithelial differentiation has a profound effect on the action of dex on the URR of HPV31, suggesting that glucocorticoids play an important role in the differentiation-dependent life cycle of HPV.     

Variant upstream regulatory region sequences differentially regulate human papillomavirus type 16 DNA replication throughout the viral life cycle

While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P(97) activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.     

Differential Requirements for Conserved E2 Binding Sites in the Life Cycle of Oncogenic Human Papillomavirus Type 31

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062–3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.     

北京地区宫颈癌HPV16上游调控序列、E6、E7癌基因序列初步分析

为检测HPV16上游调控序列(Upstream regulatory region, URR)、E6、E7癌基因变异在北京地区宫颈癌患者癌组织中的分布特征, 探讨该地区宫颈癌发生同HPV16变异株间的相关性, 文章以提取的31例HPV16检测阳性宫颈癌组织DNA为模板, 设计针对性引物扩增URR、E6、E7 3个目的片段, PCR产物直接测序并通过GenBank对比分析变异和分支鉴定情况。在所分析的宫颈癌组织中, URR是突变频率最高的片段, 其次为E7, 最保守的序列为E6。共发现热突变位点8个, 分别为URR序列上G7521A(100%)、C7435G(96.77%)、C24T(45.16%)、A7729C(45.16%)、G7839A(45.16%); E6序列上T178G(41.94%); E7序列上A647G(45.16%)、T846C(45.16%)。HPV16分支分布频率最广的是As型(54.84%), 其次为E型(45.16%)。研究结果提示, HPV16URR序列上G7521A、A7729C、G7839A, E6序列上T178G、T350G, E7序列上A647G、G658A等位点的变异可能与病毒致癌潜能及宫颈癌的发生相关。北京地区宫颈癌患者中As和E型可能是两种最主要的HPV16分支, 这有可能会为HPV疫苗的研制和感染治疗提供有价值的信息。As型和E型病毒在不同年龄组和不同肿瘤分期组的患者中分布频率有差异, 这可能会为揭示宫颈癌年轻化趋势提供新的线索。     

Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle

Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.     

Human papillomavirus type 16 E1circumflexE4 contributes to multiple facets of the papillomavirus life cycle

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1(circumflex)E4 is a viral protein that has been ascribed multiple biochemical properties of potential biological relevance to the viral life cycle. To identify the role(s) of the viral E1(circumflex)E4 protein in the HPV life cycle, we characterized the properties of HPV type 16 (HPV16) genomes harboring mutations in the E4 gene in NIKS cells, a spontaneously immortalized keratinocyte cell line that when grown in organotypic raft cultures supports the HPV life cycle. We learned that E1(circumflex)E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1(circumflex)E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1(circumflex)E4 mutant HPV16 genomes there were alterations in the frequency of suprabasal cells supporting DNA synthesis, the levels of viral DNA amplification, and the degree to which the virus perturbs differentiation. Interestingly, the comparison of the phenotypes of various mutations in E4 indicated that the E1(circumflex)E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1(circumflex)E4 is a multifunctional protein and that the different properties of E1(circumflex)E4 contribute to different processes in both the early and late stages of the virus life cycle.