scholarly journals Variant Upstream Regulatory Region Sequences Differentially Regulate Human Papillomavirus Type 16 DNA Replication throughout the Viral Life Cycle

2005 ◽  
Vol 79 (10) ◽  
pp. 5914-5922 ◽  
Author(s):  
Walter G. Hubert
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Viral Replication ◽  
Regulatory Region ◽  
Dna Amplification ◽  
Plasmid Replication ◽  
Upstream Regulatory Region ◽  
Viral Genomes ◽  
Human Papillomavirus Type 16 ◽  
Replication Factors

ABSTRACT 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 P97 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.

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

1999 ◽  
Vol 73 (3) ◽  
pp. 1835-1845 ◽  
Author(s):  
Walter G. Hubert ◽  
Taro Kanaya ◽  
Laimonis A. Laimins
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Viral Replication ◽  
Binding Site ◽  
Regulatory Region ◽  
Point Mutations ◽  
Dna Amplification ◽  
Upstream Regulatory Region ◽  
Viral Dna ◽  
Replication Factors

ABSTRACT 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.

scholarly journals Genetic Analysis of cis Regulatory Elements within the 5′ Region of the Human Papillomavirus Type 31 Upstream Regulatory Region during Different Stages of the Viral Life Cycle

2002 ◽  
Vol 76 (10) ◽  
pp. 4798-4809 ◽  
Author(s):  
Ellora Sen ◽  
Jennifer L. Bromberg-White ◽  
Craig Meyers
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Mutational Analysis ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Viral Life Cycle ◽  
Viral Gene ◽  
Upstream Regulatory Region ◽  
Kinase C

ABSTRACT The function of the 5′ region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5′ region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5′ region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.

scholarly journals Genetic and Biochemical Analysis of cis Regulatory Elements within the Keratinocyte Enhancer Region of the Human Papillomavirus Type 31 Upstream Regulatory Region during Different Stages of the Viral Life Cycle

2004 ◽  
Vol 78 (2) ◽  
pp. 612-629 ◽  
Author(s):  
Ellora Sen ◽  
Samina Alam ◽  
Craig Meyers
Keyword(s):  
Transcription Factors ◽  
Human Papillomavirus ◽  
Life Cycle ◽  
Mutational Analysis ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Viral Life Cycle ◽  
Keratinocyte Differentiation ◽  
Viral Gene ◽  
Upstream Regulatory Region

ABSTRACT Using linker scanning mutational analysis, we recently identified potential cis regulatory elements contained within the 5′ upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the viral life cycle. For the present study, we extended the linker scanning mutational analysis to identify potential cis elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the viral life cycle. The linker scanning mutational analysis identified viral cis elements located in the KE region that regulate transcription in the presence and absence of any viral gene products or viral DNA replication and determine the role of host tissue differentiation on viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same cis regulatory elements at different stages of the HPV differentiation-dependent life cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify cis regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the viral life cycle. The variable recruitment of transcription factors to the same cis element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.

scholarly journals Two Novel Promoters in the Upstream Regulatory Region of Human Papillomavirus Type 31b Are Negatively Regulated by Epithelial Differentiation

1999 ◽  
Vol 73 (4) ◽  
pp. 3505-3510 ◽  
Author(s):  
Michelle A. Ozbun ◽  
Craig Meyers
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Regulatory Region ◽  
Viral Life Cycle ◽  
Epithelial Differentiation ◽  
Upstream Regulatory Region ◽  
Organotypic Cultures

ABSTRACT Organotypic cultures support the stratification and differentiation of keratinocytes and the human papillomavirus (HPV) life cycle. We report transcription from four novel promoters in the HPV31b upstream regulatory region during the viral life cycle in organotypic cultures. Promoter initiation was not differentiation dependent; two promoters were down-regulated upon epithelial differentiation.

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

1998 ◽  
Vol 72 (2) ◽  
pp. 1071-1077 ◽  
Author(s):  
Frank Stubenrauch ◽  
Hock B. Lim ◽  
Laimonis A. Laimins
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Viral Replication ◽  
Binding Sites ◽  
Regulatory Region ◽  
Viral Life Cycle ◽  
Expression Vectors ◽  
Late Gene Expression ◽  
Fourfold Increase ◽  
Proximal Site

ABSTRACT 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.

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