scholarly journals Chaperone Proteins Abrogate Inhibition of the Human Papillomavirus (HPV) E1 Replicative Helicase by the HPV E2 Protein

2002 ◽  
Vol 22 (18) ◽  
pp. 6592-6604 ◽  
Author(s):  
Biing Yuan Lin ◽  
Alexander M. Makhov ◽  
Jack D. Griffith ◽  
Thomas R. Broker ◽  
Louise T. Chow
Keyword(s):  
Human Papillomavirus ◽  
Topoisomerase I ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Chaperone Proteins ◽  
Viral Origin ◽  
Replicative Helicase ◽  
E2 Protein ◽  
Hpv Dna ◽  
Atp Binding Site

ABSTRACT Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.

scholarly journals Dynamic Localization of the Human Papillomavirus Type 11 Origin Binding Protein E2 through Mitosis While in Association with the Spindle Apparatus

2006 ◽  
Vol 80 (10) ◽  
pp. 4792-4800 ◽  
Author(s):  
Luan D. Dao ◽  
Aaron Duffy ◽  
Brian A. Van Tine ◽  
Shwu-Yuan Wu ◽  
Cheng-Ming Chiang ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Binding Protein ◽  
Cellular Protein ◽  
Bovine Papillomavirus ◽  
Mitotic Spindles ◽  
Mitotic Chromosomes ◽  
Mitotic Apparatus ◽  
Viral Origin ◽  
E2 Protein ◽  
Origin Binding Protein

ABSTRACT Papillomaviral DNA replicates as extrachromosomal plasmids in squamous epithelium. Viral DNA must segregate equitably into daughter cells to persist in dividing basal/parabasal cells. We have previously reported that the viral origin binding protein E2 of human papillomavirus types 11 (HPV-11), 16, and 18 colocalized with the mitotic spindles. In this study, we show the localization of the HPV-11 E2 protein to be dynamic. It colocalized with the mitotic spindles during prophase and metaphase. At anaphase, it began to migrate to the central spindle microtubules, where it remained through telophase and cytokinesis. It was additionally observed in the midbody at cytokinesis. A peptide spanning residues 285 to 308 in the carboxyl-terminal domain of HPV-11 E2 (E2C) is necessary and sufficient to confer localization on the mitotic spindles. This region is conserved in HPV-11, -16, and -18 and bovine papillomavirus type 4 (BPV-4) E2 and is also required for the respective E2C to colocalize with the mitotic spindles. The E2 protein of bovine papillomavirus type 1 is tethered to the mitotic chromosomes via the cellular protein Brd4. However, the HPV-11 E2 protein did not associate with Brd4 during mitosis. Lastly, a chimeric BPV-1 E2C containing the spindle localization domain from HPV-11 E2C gained the ability to localize to the mitotic spindles, whereas the reciprocal chimera lost the ability. We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections.

Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1825-1832 ◽  
Author(s):  
Mark Stevenson ◽  
Lucy C. Hudson ◽  
Julie E. Burns ◽  
Roy L. Stewart ◽  
Michael Wells ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cervical Intraepithelial Neoplasia ◽  
Intraepithelial Neoplasia ◽  
Lower Grade ◽  
Invasive Squamous Cell Carcinoma ◽  
Hpv 16 ◽  
E2 Protein ◽  
Hpv Dna ◽  
Human Papillomavirus Type 16 ◽  
Invasive Carcinomas

The human papillomavirus type 16 (HPV-16) status of 43 cervical biopsies, which had been characterized histologically as normal, various grades of cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma, was examined by using (i) a novel antibody against the HPV-16 E2 protein, (ii) sensitive HPV-16 DNA in situ hybridization and (iii) microdissection/PCR for the E2 ORF. The data indicate that E2 protein expression is highest in koilocytes in lower-grade CIN (I), but decreases with increasing grade, whereas the detection of HPV DNA is delayed until CIN I/II, rising to the highest levels in carcinoma cells. Co-localization of E2 with HPV-16 DNA-positive cells was most commonly observed in koilocytes in CIN II lesions. PCR analyses of microdissected epithelium from the same or serial sections indicated that E2 ORFs were retained in an intact form in a number of higher-grade CIN lesions and invasive carcinomas.

scholarly journals Human Papillomavirus Type 16 E2 Protein Has No Effect on Transcription from Episomal Viral DNA

2003 ◽  
Vol 77 (3) ◽  
pp. 2021-2028 ◽  
Author(s):  
Viviane Bechtold ◽  
Peter Beard ◽  
Kenneth Raj
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Cell Lines ◽  
Trichostatin A ◽  
Viral Dna ◽  
Secondary Importance ◽  
E2 Protein ◽  
Hpv Dna ◽  
Episomal Hpv16 ◽  
E6 And E7

ABSTRACT The human papillomavirus (HPV) E2 protein plays an important role in viral DNA replication. Many studies with high-risk HPVs have demonstrated that the E2 protein can also repress transcription of the E6 and E7 oncogenes. This conclusion, based on experiments carried out with cervical cancer cells bearing integrated HPV genomes, is currently assumed to be applicable to the normal HPV life cycle, in which the viral genomes are episomal. Here, we have tested experimentally whether this assumption is correct. We made use of a pair of isogenic cell lines, W12 and S12. W12 cells contain episomal HPV16 genomes, whereas S12 cells, which are derived from the W12 line, contain HPV DNA as integrated copies. When we expressed E2 in S12 cells, we observed strong repression of E6 and E7 transcription. In contrast, no effect of E2 on the transcription of these genes was detected in W12 cells. While integration of the viral genome into the host DNA contributes to the difference between W12 and S12 cells, integration by itself is not sufficient to explain this difference. Instead, the chromatin structure in the region of the E6 and E7 promoter (p97), which we show to be very different in these two cell lines, is likely to be the cause of the different responsiveness of p97 to the E2 protein. Experiments with the histone deacetylase inhibitor trichostatin A (TSA) indicated that the episomal HPV16 DNA is in a relatively inaccessible state prior to TSA treatment. Our results, together with those of others, suggest that any effect of the E2 protein on the expression of the E6 and E7 genes during the normal viral life cycle is of secondary importance compared to the function of E2 in replication.

scholarly journals The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication

1998 ◽  
Vol 72 (4) ◽  
pp. 3436-3441 ◽  
Author(s):  
Nianxiang Zou ◽  
Jen-Sing Liu ◽  
Shu-Ru Kuo ◽  
Thomas R. Broker ◽  
Louise T. Chow
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Expression Vectors ◽  
Atpase Domain ◽  
Viral Origin ◽  
Hpv 16 ◽  
E2 Protein ◽  
Amino Terminal ◽  
E1 Protein ◽  
Carboxyl Terminal

ABSTRACT The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.

scholarly journals Human TATA Binding Protein Inhibits Human Papillomavirus Type 11 DNA Replication by Antagonizing E1-E2 Protein Complex Formation on the Viral Origin of Replication

2002 ◽  
Vol 76 (10) ◽  
pp. 5014-5023 ◽  
Author(s):  
Kelly A. Hartley ◽  
Kenneth A. Alexander
Keyword(s):  
Transcription Factors ◽  
Human Papillomavirus ◽  
Dna Replication ◽  
Dna Binding ◽  
Tata Binding Protein ◽  
Keratinocyte Differentiation ◽  
Origin Of Replication ◽  
Viral Origin ◽  
Hpv Dna

ABSTRACT The human papillomavirus (HPV) protein E2 possesses dual roles in the viral life cycle. By interacting directly with host transcription factors in basal keratinocytes, E2 promotes viral transcription. As keratinocyte differentiation progresses, E2 associates with the viral helicase, E1, to activate vegetative viral DNA replication. How E2's major role switches from transcription to replication during keratinocyte differentiation is not understood, but the presence of a TATA site near the viral origin of replication led us to hypothesize that TATA-binding protein (TBP) could affect HPV replication. Here we show that the C-terminal domain of TBP (TBPc) is a potent inhibitor of E2-stimulated HPV DNA replication in vitro (50% inhibitory concentration = 0.56 nM). Increasing the E1 concentration could not overcome TBPc inhibition in replication assays, indicating that TBPc is a noncompetitive inhibitor of E1 binding. While direct E2-TBPc association could be demonstrated, this interaction could not fully account for the mechanism of TBPc-mediated inhibition of viral replication. Because E2 supports sequence-specific binding of E1 to the viral ori, we proposed that TBPc antagonizes E1-ori association indirectly through inhibition of E2-DNA binding. Indeed, TBPc potently antagonized E2 binding to DNA in the absence (Ki = 0.5 ± 0.1 nM) and presence (Ki = 0.6 ± 0.3 nM) of E1. Since E2 and TBPc cannot be coadjacent on viral sequences, direct DNA-binding competition between TBPc and E2 was responsible for replication inhibition. Given the ability of TBPc to inhibit HPV DNA replication in vitro and data indicating that TBPc antagonized E2-ori association, we propose that transcription factors regulate HPV DNA replication as well as viral transcription.

scholarly journals Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state

2020 ◽  
Vol 48 (14) ◽  
pp. 7973-7980
Author(s):  
Ahmet C Karabulut ◽  
Ryan T Cirz ◽  
Andrew F Taylor ◽  
Gerald R Smith
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Strand Exchange ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Activated State ◽  
Complex Enzymes

Abstract Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase–nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5′-ended strand and its slower RecB helicase on the 3′-ended strand. At Chi hotspots (5′ GCTGGTGG 3′), RecB’s nuclease cuts the 3′-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB’s ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

scholarly journals Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

2020 ◽  
Author(s):  
Ahmet C. Karabulut ◽  
Ryan T. Cirz ◽  
Gerald R. Smith
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Strand Exchange ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Activated State ◽  
Complex Enzymes

ABSTRACTCoordination of multiple activities of complex enzymes is critical for life, including transcribing, replicating, and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5’-ended strand and its slower RecB helicase on the 3’-ended strand. At Chi hotspots (5’GCTGGTGG3’), RecB’s nuclease cuts the 3’-ended strand and loads RecA strand-exchange protein onto it. We report here that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate’s length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA distal end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. Computation docks NSAC1003 into the ATP-binding site, suggesting that NSAC1003 acts directly on RecB. NSAC1003 will help elucidate the molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes whose activities are coordinated at chromosomal sites.

scholarly journals Identification of Domains of the Human Papillomavirus Type 11 E1 Helicase Involved in Oligomerization and Binding to the Viral Origin

2000 ◽  
Vol 74 (16) ◽  
pp. 7349-7361 ◽  
Author(s):  
Steve Titolo ◽  
Alex Pelletier ◽  
Anne-Marie Pulichino ◽  
Karine Brault ◽  
Elizabeth Wardrop ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Papillomavirus ◽  
Dna Replication ◽  
Simian Virus 40 ◽  
Binding Domain ◽  
Atp Binding ◽  
Viral Origin ◽  
Single Stranded Dna ◽  
Terminal Domain

ABSTRACT The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by single-stranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.

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