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. 2015 Jan 15;89(2):1129-42.
doi: 10.1128/JVI.01903-14. Epub 2014 Nov 5.

A conserved regulatory module at the C terminus of the papillomavirus E1 helicase domain controls E1 helicase assembly

Stephen Schuck  1 , Arne Stenlund  2
Affiliations

A conserved regulatory module at the C terminus of the papillomavirus E1 helicase domain controls E1 helicase assembly

Stephen Schuck et al. J Virol. .

Abstract

Viruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double-stranded origin of DNA replication (ori) DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur.

Importance: This study provides a mechanistic understanding into how a multifunctional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+ helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities.

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Figures

FIG 1
FIG 1
A C-terminal 28-aa peptide is important for complex formation by E1308–605. (A) The C-terminal regions from 6 different papillomavirus E1 proteins and simian virus 40 (SV40) T-ag were aligned. Highly conserved residues are highlighted, and the acidic region is bracketed. (B) A 41-mer ssDNA probe was incubated with three levels (8, 16, and 32 ng) of E1308–605 (lanes 2 to 4) or E1308–577 (lanes 6 to 8) in the presence of ADP and subjected to EMSA. In lanes 1 and 5, no protein was added. The position of the E1 hexamer complex is indicated. (C) Two femtomoles of helicase substrate was incubated with four quantities (8, 16, 32, and 64 ng) of full-length E11–605 (lanes 3 to 6), E1308–605 (lanes 7 to 10), and E1308–577 (lanes 11 to 14) in the presence of ATP. Lane 1 contained probe alone, while lane 2 contained boiled probe. The migrations of dsDNA and ssDNA are indicated. (D) Binding reactions for EMSA with E1308–605, as shown in panel B, were scaled up and sedimented in a 15 to 30% glycerol gradient. The radioactivity in the fractions was quantitated and plotted. The peak fractions were loaded onto an EMSA gel to verify that the gradient peak corresponded to the complex that we observe by EMSA (inset). Arrows indicate the sedimentation of the marker proteins bovine serum albumin (BSA), alcohol dehydrogenase (ADH), and β-amylase. (E) Binding reactions for EMSA with E1308–577, as shown in panel B, were scaled up and sedimented in a 15 to 30% glycerol gradient. The radioactivity in the fractions was quantitated and plotted. The last fraction plotted corresponds to the bottom portion of the tube, which was cut off and quantified. (F) One hundred micrograms of E1308–577 was sedimented in a 5 to 30% glycerol gradient. The protein was detected by Bradford assays and compared to the sedimentation of the marker proteins carbonic anhydrase and BSA.
FIG 2
FIG 2
The C-terminal 28 residues regulate E1 hexamer formation. (A) Cartoon showing the sequence of the C-terminal module with deletions and point mutations in the context of E308–605. (B) The deletion mutants in the C-terminal module were tested for hexamer formation in the context of E1308–605 using an ssDNA probe. Two, 4, and 8 ng each of the proteins E1308–577, E1308–584, E1308–589, E1308–592, E1308–597, E1308–600, and E1308–605 were incubated with a 41-mer ssDNA probe (∼2 fmol) in the presence of ADP and tested for hexamer formation by EMSA. The migration of the E1 hexamer is indicated. (C) Two, 4, and 8 ng each of the proteins with point mutations G588A, F594A, and C596A and the deletion mutants E1308–577 7×A and E1308–577 7×N were incubated with a 41-mer ssDNA probe in the presence of ADP and tested for hexamer formation by EMSA. (D) One hundred micrograms of E1308–592 was sedimented in a 5 to 30% glycerol gradient. The protein was detected by Bradford assays and its sedimentation was compared to that of the marker proteins carbonic anhydrase and bovine serum albumin (BSA).
FIG 3
FIG 3
Mutations in the C-terminal module affect the helicase and ATPase activity of E1. A helicase substrate was generated by annealing a primer to M13 ssDNA followed by extension with a mixture of deoxy- and dideoxynucleoside triphosphates (dNTPs and ddNTPs) and Klenow DNA polymerase to generate partially double-stranded templates with long (0.2- to 5-kb) radiolabeled strands. In each set, 2 fmol of the substrate was incubated with 3 levels (4, 8, and 16 ng) of the proteins. Proteins E1308–577, E1308–584, E1308–589, and E1308–592 (A), E1308–597, E1308–600, and E1308–605 (B), E1308–577, E1308–577 7×A, E1308–577 7×N, and E1308–605 (C), and E1308–605, E1308–605 G588A, E1308–605 F594A, and E1308–605 C596A (D) were tested. In each panel, a size marker (first lane), a boiled probe (B), and a no-E1 (−) sample were included. (E) Point mutants and deletions in the C-terminal module in the context of E1308–605 were tested for ATPase activity. Samples were incubated with [γ-32P]ATP in the presence of ssDNA except where otherwise indicated, followed by separation of free phosphate from ATP by thin-layer chromatography. The level of hydrolysis is shown below each lane.
FIG 4
FIG 4
The acidic region is required for DT formation, and the C-terminal tail is required for DH formation by E11–605. (A) Cartoon showing the deletions and point mutations in the C-terminal module in the context of E11–605. (B) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of the deletion mutants E11–597, E11–592, E11–589, E11–584, and E11–577 in the presence of ADP and tested for DT formation by EMSA. The mobility of the DT complex is indicated. (C) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of the mutants E11–577 7 × N, E11–577, E11–605 G588A, E11–605 M591A, E11–605 T593A, E11–605 F594A, and E11–605 C596A in the presence of ADP and tested for DT formation. The mobility of the DT complex is indicated. (D) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of the mutants E11–577 7×N, E11–577, E11–584, E11–589, E11–592, E11–597, and E11–600 in the presence of ATP and tested for DH formation. The mobility of the DH complex is indicated. (E) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of the mutant proteins E11–577 7×A, E11–577, E11–605 G588A, E11–605 M591A, E11–605 T593A, E11–605 F594A, and E11–605 C596A and tested for DH formation in the presence of ATP by EMSA. (F) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of wt E1 or the mutants E11–605 579R/583R and E11–577 and tested for DT formation in the presence of ADP by EMSA. For the mutants E11–605 586R, E11–605 579R/583R/586R, and E11–605 579R/583R/586R/587R, four levels of E1 (1.2, 2.5, 5 and 10 ng) were used. (G) To determine whether the arginine substitutions in the acidic region caused structural defects, these mutants were tested for the ability to form the E12E22 complex: 1.2 and 2.5 ng of wt E1 or the mutant E11–605 586R, E11–605 579R/583R, E11–605 579R/583R/586R, or E11–605 579R/583R/586R/587R were incubated with the 84-bp ori probe and 2 ng of E2 and tested for E12E22 complex formation by EMSA. In lane 12, 1.2 ng of E11–577 was tested for E12E22 complex formation.
FIG 5
FIG 5
The C-terminal module is required for unwinding and DNA replication in vitro. (A) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of E11–584, E11–589, E11–592, E11–597, E11–600, and E11–605 in the presence of ATP and E. coli SSB and analyzed by EMSA. ssDNA + SSB complexes are indicated. (B) The 84-bp ori probe was incubated with three levels (2.5, 5, and 10 ng) of E11–605, E11–605 G588A, E11–605 M591A, E11–605 T593A, E11–605 F594A, E11–605 C596A, and E11–577 7×N in the presence of ATP and E. coli SSB and analyzed by EMSA. (C) The 84-bp ori probe was incubated with four levels (2.5, 5, 10 and 20 ng) of E11–605, E11–605 586R, E11–605 579R/583R, E11–605 579R/583R/586R, and E11–577 in the presence of ATP and E. coli SSB and analyzed by EMSA. (D) Low levels of Sarkosyl disrupt E1 complexes with ssDNA and dsDNA but do not affect complexes between E. coli SSB and ssDNA. The double-stranded 84-bp ori probe was incubated with E1 in the presence of ADP (lanes 2 and 3). Immediately before loading onto an EMSA gel, one sample (lane 3) was treated with 0.1% Sarkosyl. The ssDNA probe was incubated with E1 in the presence of ADP (lanes 5 and 6). Immediately before loading onto an EMSA gel, one sample (lane 6) was treated with 0.1% Sarkosyl. The ssDNA probe was incubated with E. coli SSB (lanes 7 and 8). Immediately prior to loading onto the EMSA gel, one sample (lane 8) was treated with 0.1% Sarkosyl. (E) Deletions and point mutations in the acidic region and C tail were expressed and purified from E. coli and tested for DNA replication in vitro as described in Materials and Methods. Two hundred nanograms of each mutant protein was incubated in replication extract in the presence of [α-32P]dCTP and analyzed by agarose gel electrophoresis. (F) The arginine substitutions in the acidic region were tested for DNA replication in vitro. Three quantities (100, 200, and 400 ng) of wt E1 and the E1 579R/583R, 586R, 579R/583R/586R mutants and E11–577 were used in cell-free replication reactions in the presence of [α-32P]dCTP and analyzed by agarose gel electrophoresis. (G) Inactivation of the acidic region in the context of the E1308–605 C596A restores E1 hexamer formation and ATPase activity to wt levels. Mutants in the C-terminal module were tested for hexamer formation in the context of E1308–605 using an ssDNA probe. Two, 4, and 8 ng each of the proteins E1308–605, E1308–605 C596A, and E1308–605 C596A D579R/D573RE586R were incubated with a 41-mer ssDNA probe (∼2 fmol) in the presence of ADP and tested for hexamer formation by EMSA. The migration of the E1 hexamer is indicated. The altered mobility of the complexes formed with E1308–605 C596A D579R/D573RE586R is caused by the E586R substitution. (H) Mutants in the C-terminal module in the context of E1308–605 were tested for ATPase activity. The proteins E1308–605, E1308–605 C596A, andE1308–605 C596A D579R/D573RE586R were incubated with [γ-32P]ATP and ssDNA, unless otherwise indicated, and tested for ATPase activity as described in Materials and Methods. The migration of ATP and free phosphate is indicated, and the level of hydrolyzed ATP is shown below the lanes.
FIG 6
FIG 6
The C-terminal module interacts with the E1 oligomerization domain. (A) E1308–577 and luciferase were translated in vitro in a reticulocyte lysate in the presence of [35S]methionine, used in pulldown experiments with GST, GST544–577 579R/583R/586R, or GST544–605, and analyzed by SDS-PAGE followed by autoradiography. In the input lane, 1% of input was loaded. (B) The same gel as that described for panel A was stained with Coomassie brilliant blue (CBB) to show the presence of the GST fusion proteins. (C) E1308–410 was translated in vitro in a reticulocyte lysate in the presence of [35S]methionine, used in pulldown experiments with GST, GST544–577 579R/583R/586R, or GST544–577, and analyzed by SDS-PAGE followed by autoradiography. (D) The same gel as for panel C was stained with CBB to show the presence of the GST fusion proteins. (E) The C tail is required to sense the presence of ssDNA. Three quantities (2.5, 5, or 10 ng) of wt E1 or the F594A or C596A mutant were used in EMSA using two different probes. Wild-type E1 was incubated with the wt 84-bp ori probe in the presence of ADP or ATP. Wild-type E1 or the F594A or C596A mutant was incubated with a probe that differed from the wt probe by a 6-bp mismatched region (bubble probe) in the presence of ADP. Migration of the DT and DH complexes is indicated.
FIG 7
FIG 7
Effects of mutations in the C-terminal module on E1 complex formation and model of control of oligomerization of E1 by the C-terminal module. (A) Summary of how point mutations and deletions in the C-terminal module affect hexamer (H), double trimer (DT), and double hexamer (DH) formation in the context of E1308–605 and E11–605. (B) Model for how the acidic region and C tail control oligomerization of E1. See the text for details.
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