The carboxyl-terminal region of the human papillomavirus type 16 E1 protein determines E2 protein specificity during DNA replication - PubMed (original) (raw)

The carboxyl-terminal region of the human papillomavirus type 16 E1 protein determines E2 protein specificity during DNA replication

N Zou et al. J Virol. 1998 Apr.

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.

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Figures

FIG. 1

FIG. 1

Transient replication by combinations of HPV-11 and HPV-16 E1 and E2 proteins. Replication assays of an HPV-11 ori plasmid (nt 7730 to 7933/1 to 99) (lanes 1 to 8) or an HPV-16 ori plasmid (nt 7455 to 7906/1 to 111) (lanes 9 to 12) were conducted with human 293 cells as described in the text. The E1 and E2 expression vector plasmids used are indicated above each pair of lanes. Low-molecular-weight DNA was harvested 48 h posttransfection. Half of the recovered DNA from one 100-mm plate was subjected to _Hin_dIII and _Dpn_I double digestion (DpnI +). The other half was subjected to _Hin_dIII digestion, which linearized the three plasmids (DpnI −). The products were separated electrophoretically in a 0.8% agarose gel and transferred to a nitrocellulose membrane. The membrane was probed with the [α-32P]dCTP-labeled, origin-containing plasmid and exposed to X-ray film overnight. Only replicated DNA was resistant to _Dpn_I digestion (marked Ori), whereas the unreplicated DNA was digested to small fragments and migrated ahead of the linearized, newly replicated ori plasmid (see Fig. 3 and 4).

FIG. 2

FIG. 2

Structures, functional domains, and relative replication activities of wild-type and hybrid HPV E1 proteins. (A) Schematic representations of wild-type and hybrid E1 proteins and relative replication activities. The shaded boxes represent regions derived from HPV-16 E1, and the open boxes represent those derived from HPV-11 E1. To minimize replication differences that might be attributable to mRNA translation efficiencies, the 5′ untranslated sequence and first 5 codons of HPV-11 and of 11/16 hybrid proteins were all replaced with those from HPV-16 E1 and are denoted with an asterisk in the designation. This modification resulted in a net change of two amino acids. The BPV-1 E1 protein is shown as a thick line. Numbers refer to amino acid residues. Thin solid lines represent the BPV-1 E1 protein domains defined to be involved in E2 interactions (lines c and d) or DNA binding (line a), based on protein interaction assays in vitro or in the yeast two-hybrid system (1, 18, 23, 32, 41), or the ATPase domain (line b) based on the homology to the SV40 T antigen and mutagenic analysis (8, 26, 28). The dotted line (e) approximates the E1 domain of HPV-16 and HPV-33 involved in interaction with the E2 protein (29, 45). The dashed line (f) signifies the domain of HPV-16 E1 responsible for E2 specificity during replication (this study). After subtraction of background signals, the relative replication activities (Rel Activity) of each E1 protein in the presence of HPV-11 or HPV-16 E2 protein were determined by PhosphorImager quantification of the results shown in Fig. 3 and 4A. The results are shown to the right of each clone. The activities achieved with HPV-16 E1 and E2 were taken as 100% and are assigned a score of ++++. Scores: −, relative activity of less than 5%, including signals detectable only after a long exposure; +, activity between 5 and 10%; ++, activity between 10 and 50%; +++, activity between 50 and 90%; ++++, activity over 90%. (B) Peptide sequences spanning the junctions of hybrid E1 proteins. The numbers flanking the arrows are amino acid residues where protein coding switches from one virus type to the other.

FIG. 3

FIG. 3

Transient replication of an HPV-11 ori plasmid by HPV-16/11 E1 hybrid proteins in combination with either HPV-16 or HPV-11 E2 protein. Replication assays were conducted as described in the legend to Fig. 1. The low-molecular-weight DNA was digested overnight with both _Dpn_I and _Hin_dIII. Fragments that migrated faster than the linearized ori plasmid were derived from _Dpn_I-sensitive input DNA. Similar results were obtained when an HPV-16 ori plasmid was used (data not shown).

FIG. 4

FIG. 4

Transient replication by HPV-11*/16 E1 hybrid proteins in combination with either HPV-16 or HPV-11 E2 protein. (A) Replication of an HPV-11 ori plasmid; (B) replication of an HPV-16 ori plasmid.

FIG. 5

FIG. 5

Expression of wild-type and HPV-16/11 hybrid E1 proteins in COS-7 cells. The EE-epitope-tagged wild-type and hybrid E1 proteins were each expressed from the pMT2 vector. After 24 h of incubation, the transfected cells were disrupted in 200 μl of lysis buffer. Forty microliters of each lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The E1 proteins were detected by monoclonal antibody to the EE epitope (14). The rightmost lane contains EE-epitope-tagged HPV-16 E1 protein purified from insect Sf9 cells infected with a recombinant baculovirus (21).

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