Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1

Sun, Shaw; Thorner, Lauren; Lentz, Michael; MacPherson, Paul; Botchan, Michael R.

doi:10.1128/jvi.64.10.5093-5105.1990

Cited by 86 publications

(52 citation statements)

References 54 publications

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“…Amino acid similarities suggested that El, like SV40 large-T antigen, may be a DNA helicase/ATPase involved in initiating viral DNA replication (3). Subsequently, BPV-1 El protein was shown to be a nuclear 68-72 kD ATP-binding phosphoprotein (4,5,6). A mutation in El (Pro-434 to Ser), analogous to that in the SV40 large-T antigen mutant tsA209 that impairs ATPbinding, renders BPV-1 defective for DNA replication and abolishes ATP-binding by El (6).…”

Section: Introductionmentioning

confidence: 99%

E1 protein of human papillomavirus is a DNA helicase/ATPase

1993

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Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2. Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein. However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems. We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E. coli as a maltose-binding protein fusion (MBP-E1), for the first time. As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E. coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities. We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker. This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1. We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain. HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.

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Section: Introductionmentioning

confidence: 99%

E1 protein of human papillomavirus is a DNA helicase/ATPase

1993

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“…The nuclear-localised early protein E l shares some biochemical characteristics with SV40 large T antigen, including helicase and ATPase activities (Clertant and Seif, 1984;Sun et al, 1990). The SV40 large T antigen is the only viral protein required for SV40 replication (Myers and Tjian, 1980) and the functions of the E l protein are consistent with it performing a similar role.…”

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confidence: 98%

The Interaction between Human Papillomavirus Type 16 El and E2 Proteins is Blocked by An Antibody to the N‐Terminal Region of E2

Hibma

Raj

Ely

et al. 1995

European Journal of Biochemistry

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El could be blocked by a monoclonal antibody that bound E2 in the region of amino acids 18-41 of E2 whereas a monoclonal antibody reactive with a nearby part of the molecule (amino acids 2-17) only partially blocked this interaction. These results suggest that a region in the N-terminus of E2 around amino acids 18-41 is a site of interaction with the E l protein.Keywords. Papillomavirus; HPV16; E l ; E2; protein interactions.The papillomaviruses are a group of over 70 types of doublestranded DNA viruses that can infect a variety of animal species. Viral infection is associated with the formation of benign epithelial tumours and, depending on the viral type, has been linked with malignant carcinoma. Human papillomavirus type 16 (HPV16) is a high risk type that is frequently associated with progression to malignancy in the human cervix (zur Hausen and Schneider, 1987).A number of early open reading frames have been defined for HPV16. The gene product of the E2 open reading frame is a 365-amino-acid protein, divided into three functional domains. The N-terminal trans-acting domain is separated from a C-terminal DNA-binding domain by a proline-rich hinge (Gin and Yaniv, 1988).The E2 protein has an important regulatory function as a trans-acting protein in the control of papillomavirus P,, promoter. This promoter regulates expression of HPV16 E6 and E7 proteins, which are involved in cellular transformation (Munger et al., 1992). Although HPV16 E2 can trans-activate heterologous promoters such as the simian virus 40 (SV40) early promoter, it has a trans-repressing effect on the HPV16 P , promoter. This promoter region contains several sites for cellular trans-activators such as Spl and AP1 and is activated in the absence of E2 (Cripe et al., 1990). The repressive effect of E2 on the P,, promoter is likely to be due, at least in part, to E2 DNA binding causing displacement of other transcription factors sucfi as Spl and TFIID from the promoter region (Tan et al., 1992(Tan et al., , 1994.The replicative function of the E2 gene product has been extensively studied in bovine papillomavirus type 1 (BPVl), as DNA from this type can become established as a stable, replicated plasmid in murine and bovine fibroblasts (Lambert, 1991). The ability of viral DNA to replicate in this system is dependent on the presence of the gene products of the viral E l and E2 open reading frames (OW) in association with other cellular factors (DiMaio and Settleman, 1988). Similarly, replication of HPVll DNA in a squamous carcinoma cell line requires the presence of both E l and E2. Using the same system, it has been possible to replicate the origin of HPV16 but only in the presence of HPVll E l and HPV16 E2 expressed from strong heterologous promoters (del Vecchio et al., 1992).The nuclear-localised early protein E l shares some biochemical characteristics with SV40 large T antigen, including helicase and ATPase activities (Clertant and Seif, 1984;Sun et al., 1990). The SV40 large T antigen is the only viral protein required for SV40 replicati...

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“…It is therefore likely that the differences between SV40 and BPV1 lie with the proteins involved in the initiation of replication. The E1 protein, which shares a number of biochemical properties with the T antigen, has been clearly established as the initiator of BPV1 replication (33,35,36,43,52). The role of the E2 transactivator protein in BPV1 replication is less clear.…”

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Bovine papillomavirus E1 protein binds specifically DNA polymerase alpha but not replication protein A

Bonne-Andréa

Santucci

Clertant

et al. 1995

J Virol

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Extracts prepared from either mouse cells or monkey cells were examined for the ability to support in vitro bovine papillomavirus type 1 (BPV1) DNA replication, and they were used in parallel as a source of host replication proteins for affinity chromatography. DNA synthesis exhibited an absolute requirement for BPV1 E1 protein. In contrast to previous observations, we found that low levels of E1 were highly efficient in initiating DNA replication in the absence of the BPV1 transcription factor E2. Surprisingly, COS-1 cell extract allowed a high rate of BPV1 DNA replication, supporting an efficient production of mature circular DNA molecules, whereas in mouse cell extracts, the replication products mostly consisted of replicative intermediates. Submitting the extracts to affinity chromatography allowed specific binding of DNA polymerase ␣-primase to E1 protein, up to a total depletion of the extract, regardless of the origin of the cell extract. Furthermore, replication protein A was not retained on E1 affinity columns, even when E2 was complexed with E1. These data confirm that the interactions between E1 and DNA polymerase ␣-primase do not exhibit cell-type specificity, as had already been suggested by data from in vivo and in vitro replication assays, but they imply that other cellular proteins may affect the level of E1-dependent replication.

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Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1

Cited by 86 publications

References 54 publications

E1 protein of human papillomavirus is a DNA helicase/ATPase

E1 protein of human papillomavirus is a DNA helicase/ATPase

The Interaction between Human Papillomavirus Type 16 El and E2 Proteins is Blocked by An Antibody to the N‐Terminal Region of E2

Bovine papillomavirus E1 protein binds specifically DNA polymerase alpha but not replication protein A

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