Báo cáo khoa học: Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase ppt

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase Richard W.. Smith1,* and Heinz-Peter Nasheuer1,2 1 Abteilung Biochemie, Institut fu¨r Molekula

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase

Richard W P Smith1,* and Heinz-Peter Nasheuer1,2

1

Abteilung Biochemie, Institut fu¨r Molekulare Biotechnologie, Jena, Germany;2National University of Ireland, Galway,

Department of Biochemistry, Galway, Ireland

Host species specificity of the polyomaviruses simian virus

40 (SV40) and mouse polyomavirus (PyV) has been shown

to be determined by the host DNA polymerase a-primase

complex involved in the initiation of both viral and host

DNA replication Here we demonstrate that DNA

repli-cation of the related human pathogenic polyomavirus JC

virus (JCV) can be supported in vitro by DNA polymerase

a-primase of either human or murine origin indicating that

the mechanism of its strict species specificity differs from that of SV40 and PyV Our results indicate that this may

be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex

Keywords: DNA replication; initiation; DNA polymerase a-primase; species specificity; polyomavirus

Polyomavirus DNA replication has served as a model

system to study eukaryotic DNA replication [1,2] JC virus

(JCV) belongs to the polyomavirus family and is the

causative agent of progressive multifocal

leukoencephalo-pathy in immunocompromised humans (reviewed in [3–7])

JCV exhibits a highly restricted host range and this species

specificity appears to be governed by host encoded DNA

replication factors as hamster glial cells, which support viral

early gene transcription, nevertheless fail to replicate JCV

DNA [8] JCV is closely related to simian virus 40 (SV40)

and to mouse polyomavirus (PyV), both of which show

clear species specificities as lytic infection is limited to

primate and mouse cells, respectively [9] The species

specificities of both SV40 and PyV are regulated at the

level of initiation of DNA replication [10], a process that

has been extensively studied both in vivo and in vitro owing

to the development of cell-free DNA replication systems

[2,11–16]

Polyomavirus DNA replication is carried out by the host

cell machinery supplemented with a single essential viral

protein, large T antigen (TAg), which recognizes and

partially unwinds the viral replication origin, recruits host

proteins such as replication protein A (RPA) and DNA

polymerase a-primase, and functions as the replicative

helicase [2,17,18] Species specificity of both SV40 and PyV DNA replication can be reproduced in vitro using DNA carrying the viral core origin and purified replication enzymes [14,19–21] For both SV40 and PyV it has been clearly demonstrated that the host factor responsible for species specificity is DNA polymerase a-primase, which initiates DNA replication in all eukaryotes [19,22–27] DNA polymerase a-primase consists of four subunits with apparent molecular masses of 180, 68, 58 and 48 kDa of which the largest and smallest subunits are a DNA polymerase and a primase, respectively [28–30] SV40 DNA replication in vitro was recently shown to require a functional interaction between the SV40 TAg and the C-terminus of the p180 subunit of human DNA poly-merase a-primase [26]

The genome of JCV is 69% homologous to that of SV40 and expresses an analogous set of proteins [31] The core origins of replication of the two viruses are also conserved to such an extent that SV40 TAg, which is 72% identical to JCV TAg, can efficiently support JCV DNA replication

in vivoand in vitro [32,33] The high level of conservation between these two primate specific viruses coupled with the fact that JCV DNA replication is inhibited in a nonpermis-sive host in vivo would imply that the restricted host range of JCV is due to a requirement for human DNA polymerase a-primase, as is the case with SV40 [8] Previously we reported the establishment of a cell-free system for JCV DNA replication [32] With this system we were able to reproduce many features of JCV DNA replication found

in vivo, such as sequence requirements at the origin of replication and the requirement for JCV or SV40 (but not PyV) TAg for efficient replication Therefore, we applied this system to the question of host specificity regulation and found that this differs from that of SV40 in that it is not determined at the level of initiation of DNA replication by DNA polymerase a-primase in vitro This appears to be due

to differences in the interaction of the JCV and SV40 large

T antigens with the initiation complex

Correspondence to H P Nasheuer, National University of Ireland,

Galway, Department of Biochemistry, Cell Cycle Control

Laboratory, Galway, Ireland.

Fax: + 353 91 512 504, Tel.: + 353 91 512 409,

E-mail: h.nasheuer@nuigalway.ie

Abbreviations: JCV, JC virus; SV40, simian virus 40; PyV, mouse

polyomavirus; TAg, large T antigen; RPA, replication protein A.

*Present address: Institute of Virology, University of Glasgow,

Church Street, Glasgow G11 5JR, Scotland UK.

(Received 30 October 2002, revised 4 March 2003,

accepted 17 March 2003)

[22,32,34,35] Human and murine DNA polymerase

a-primase were immunopurified using the monoclonal

antibodies SJK237-71 and SJK287-38, respectively [36]

Human RPA was bacterially expressed and purified as

outlined previously [37,38] Human topoisomerase I was

expressed in insect cells and purified as described by Søe

et al [39] and was a generous gift of K Søe (IMB-Jena,

Germany) The monoclonal antibodies SJK237-71 and

SJK287-38, specific for DNA polymerase a-primase, were

purified by affinity chromatography [40] Protein

concen-tration was determined according to Bradford [41] using a

commercial reagent with BSA as a standard (Biorad,

Munich) DNA polymerase a and DNA primase assays

were performed as previously described [34,42,43]

Preparation of S100 extracts and replication

of SV40 and JCVin vitro

S100 extracts were prepared from logarithmically growing

FM3A cells as previously described [27,34] Cells were

harvested by centrifugation, then washed twice with

phos-phate buffered saline (NaCl/Pi) and once with hypotonic

buffer The cells were resuspended in hypotonic buffer,

incubated for 10 min on ice, and broken by 12 strokes in a

Dounce homogenizer The extracts were centrifuged at 4C

and 11 000 g The supernatant was then adjusted to

100 mMNaCl and clarified by a second centrifugation at

100 000 g (S100 extract) Depletion of DNA polymerase

a-primase from S100 extracts was performed essentially as

previously described [22,27,32]

The replication of SV40 and JCV DNA in vitro was

performed as previously described [22,27,32] Briefly, the

assay contained 0.6 lg SV40 or JCV TAg, 250 ng of

pUC-HS or pJC389 or pJC433 DNA (carrying the replication

origin of SV40 or JCV, respectively [21,32]), and 200 lg

S100 in 30 mMHepes/NaOH (pH 7.8), 1 mMdithiothreitol,

7 mM magnesium acetate, 1 mM EGTA (pH 7.8), 4 mM

ATP, 0.3 mM CTP, GTP, and UTP, 0.1 mM dATP and

dGTP, 0.05 mM dCTP and dTTP, 40 mMcreatine

phos-phate, and 80 lgÆmL)1creatine kinase, and 5 lCi each of

[a32P]dCTP and [a32P]dTTP (3000 CiÆmmol)1,

Amersham-Biosciences) DNA polymerase a-primase was added as

indicated The incorporation of radioactive dNMP was

measured by acid-precipitation of DNA and scintillation

counting The total radioactivity was measured after

spotting 5 lL of a 200-fold dilution of the replication assay

onto GF52 filters (Schleicher & Schu¨ll, Dassel, Germany)

EcoRI and DpnI digestion of product DNA was carried out

as described by Kautz et al [23]

Initiation of replication on JCV DNA

Initiation reactions were performed essentially as previously

described [22,32,44,45] Briefly, the JCV initiation assay

(40 lL) was assembled on ice and contained 0.25 lg

Brussels) Recombinant DNA polymerase a-primase was added as indicated in the figure legends After incubation for

2 h at 37C one-eighth of the reaction mixture was spotted onto DE81 paper to estimate the amount of incorporated nucleotides [46] For size analysis the reaction products were precipitated with 0.8 M LiCl, 10 lg of sonicated salmon sperm DNA (Sigma), 10 mMMgCl2and 120 lL of ethanol for 1 h on dry ice, washed twice with 75% ethanol/water, dried, redissolved in 45% formamide/5 mMEDTA/0.05% (w/v) xylene cyanol FF/0.05% (w/v) bromphenol blue at

65C for 30 min, heated for 3 min at 95 C, and electro-phoresed in denaturing 20% polyacrylamide gels for 3–4 h

at 600 V as described previously [22] The reaction products were visualized by autoradiography and quantified with a phosphoimager (Amersham Biosciences)

JCV and SV40 monopolymerase systems These assays were adapted from Ishimi et al [11] The monopolymerase assay (40 lL) was assembled on ice and contained 0.5 lg pUC-HS (carrying the SV40 replication origin) or 0.5 lg pJC433 (carrying the JCV replication origin [32]), 1 lg SV40 or JCV T antigen, 1.4 ng topoiso-merase I and 0.5 lg RPA, in 30 mMHepes/KOH (pH 7.8),

7 mM magnesium acetate, 0.1 mM EGTA, 1 mM dithio-threitol, 0.2 mMUTP, 0.2 mM GTP, 0.2 mM CTP, 4 mM ATP, 20 mM dATP, 20 mM dGTP, 2 mM dCTP, 2 mM dTTP, 40 mM creatine phosphate, 1 lg creatine kinase, 0.25 mgÆmL)1 heat treated BSA, and 5 lCi [a-32P]dCTP and 5 lCi [a-32P]dTTP (each 3000 CiÆmmol)1, Amersham-Biosciences) Recombinant DNA polymerase a-primase was added as indicated After incubation for 90 min at

37C, one-quarter of the reaction mixture was used to determine the level of incorporation by spotting onto DE81 paper, washing with 0.5MNaHCO3and liquid scintillation counting [46]

Results

Replication of JCV DNA in crude cell extracts with recombinant human and murine DNA polymerase a-primase

Previously we reported the establishment of in vitro systems for the replication of JCV DNA, either using crude cell extracts or purified proteins only [32] Both these systems were dependent upon recombinant JCV TAg and the presence of a JCV origin of DNA replication Here we applied these systems to study the dependence of JCV DNA replication on human replication proteins Figure 1 repre-sents a comparison between the SV40 (panel A) and JCV (panel B) cell-free DNA replication systems Figure 1B shows a DNA replication assay using mouse FM3A S100 crude cell extracts depleted of DNA polymerase a-primase supplemented with JCV TAg, a plasmid carrying the JCV

replication origin (pJC389) and either recombinant human

or murine DNA polymerase a-primase expressed using the

baculovirus system (Fig 1B, columns 1–7) The replication

activity of JCV TAg in mouse cell extracts is not dependent

on the sequence of the plasmid as the incorporation of

radioactive dNMPs was the same whether the plasmid

pJC389 or pJC433 was used in the cell-free replication assay

(data not shown)

In parallel, we show an SV40 DNA replication assay

using the same cell extracts but with SV40 TAg and a

plasmid (pUC-HS) that carries the SV40 replication origin

(Fig 1A, columns 1–7 [26,27]) As we showed previously,

SV40 DNA replication is absolutely dependent upon human

DNA polymerase a-primase (Fig 1A, columns 1–5 [26,27])

and more specifically, upon the human p180 subunit as the

hybrid DNA polymerase a-primase complex consisting of

the murine p180 subunit together with the human p68, p58

and p48 subunits (MH3) is inactive (Fig 1A, columns 6–7

[26,27]) In contrast, replication of JCV DNA does not show

the same strict requirement for human DNA polymerase

a-primase (Fig 1B, columns 1–7) Both murine DNA

polymerase a-primase and the hybrid complex, MH3, show

significant activity in the DNA replication assay In both the

SV40 and JCV assays, incorporation of nucleotides is

dependent upon an intact origin of replication; either

plasmids carrying the noncognate PyV origin or a disabled

JCV origin are inactive (Fig 1A, column 8 and Fig 1B,

columns 8 and 9) Background values determined without

added DNA polymerase a-primase were higher in the JCV

system, presumably due to residual endogenous murine enzyme in the cell extracts due to incomplete depletion However, as this background incorporation did not repre-sent full rounds of replication in either system (Fig 2A,B, columns 1–2) other relevant values (Fig 1A,B, lanes 2–7) were corrected for it

We further characterized the products of the replication reactions by digesting the resulting DNA with the restriction enzyme DpnI, which digests only fully methylated DNA The plasmid DNA used as template in our assay was purified from Escherichia coli and is therefore fully methy-lated However, one or more full rounds of replication will result in hemimethylated or unmethylated products and will consequently lead to DpnI resistance which is indeed observed after replication of JCV DNA either with human

or with murine DNA polymerase a-primase (Fig 2B, lanes

4 and 6) The lack of species specificity we observed was reproducible with various independently expressed and purified batches of JCV TAg (data not shown) and with various batches of the template DNAs pJC389 and pJC433 ([32]; Figs 1 and 2) As expected murine DNA polymerase a-primase did not support SV40 DNA replication (Fig 2A, lanes 5 and 6)

JCV DNA replication with purified proteins

We note that in the cell-free system JCV DNA replication is markedly less efficient when driven by murine compared with human DNA polymerase a-primase In order to

Fig 1 DNA replication assays in murine FM3A cell extracts depleted of DNA polymerase a-primase using the SV40 (A) and JCV (B) systems (A) The SV40 system makes use of SV40 TAg and pUC-HS template DNA containing the SV40 origin of DNA replication (B) The JCV system uses JCV TAg and pJC389 DNA with the JCV origin The cell extracts were supplemented with 0.5 and 1.0 DNA polymerase units of the indicated DNA polymerase a-primase complexes (H 4 , human heterotetramer; M 4 , murine heterotetramer; MH 3 , murine p180 with human p68, p58 and p48) Enzyme activities were determined beforehand with a DNA polymerase assay on activated calf thymus DNA (A) and (B) column 1, TAg omitted; column 8, pJC389I-/II-, containing a disabled JCV replication origin [32], was used as template for human DNA polymerase a-primase with SV40 (A) and JCV TAg (B), respectively (B) column 9, pUC-Py1, containing the PyV replication origin [21], used with human DNA polymerase a-primase and JCV TAg Values in panels (A) and (B), columns 2–7 were corrected for background incorporation determined in either system without the addition of DNA polymerase a-primase Standard deviations from two to four experiments are indicated as error bars The experiments

in (A) and (B) were performed in parallel.

characterize further these processes we made use of purified systems Firstly we examined the efficiency of primer formation at the origin of replication during the initiation step of JCV DNA replication Figure 3 shows that this is efficiently carried out by both the human and murine DNA polymerase a-primase complexes although quantification of the reaction products shows the murine complex to be approximately 30% less efficient than the human complex, especially in the synthesis of products greater than four nucleotides in length Murine DNA polymerase a-primase

is absolutely inactive in initiating SV40 DNA replication in

an assay consisting of purified enzymes [26,27]

We then reproduced the absence of JCV species specifi-city using the monopolymerase DNA replication system, which makes use of purified enzymes only and allows measurement of deoxyribonucleotide incorporation after coupled initiation and elongation by DNA polymerase a-primase [11] Figure 4 shows that SV40 DNA replication

in this system is clearly dependent upon DNA polymerase a-primase being of human origin (panel A) but that this is not the case for JCV (panel B) This shows that the ability of murine DNA polymerase a-primase to replicate JCV DNA

is not dependent upon factors other than the replication proteins used in this assay However, overall deoxynucleo-tide incorporation is less efficient than by human DNA polymerase a-primase as shown above with the cell-free assay (Fig 1), which suggests that steps in JCV DNA replication subsequent to primer formation may be slightly inhibited by the murine enzyme complex

SV40 TAg confers species specificity to JCV origin dependent DNA replication

It has been reported that SV40 TAg is capable of supporting JCV DNA replication both in vivo and in vitro [32,33,47] Therefore, we asked whether substitution of SV40 TAg for JCV TAg would render JCV DNA replication species-specific with regard to the nature of the DNA polymerase a-primase complex catalysing the reaction, as is the case

Fig 2 DNA synthesis products of the SV40

and JCV DNA replication systems Murine

FM3A cell extracts depleted of DNA

poly-merase a-primase were supplemented with 1.0

D NA polymerase units of H4 or M4 or not

supplemented (–Pol) One-quarter of the

DNA synthesis products were analysed for

complete DNA replication by digestion with

EcoRI and DpnI (even numbered lanes) In

parallel, the products were linearized with

EcoRI (odd numbered lanes) The positions

of linearized template DNAs are indicated

by arrows.

Fig 3 Autoradiogram of an in vitro JCV DNA replication initiation

assay with 0.2 and 0.4 units of primase of either human (H4) or murine

(M4) DNA polymerase a-primase complexes Specific primase activities

were determined beforehand with a primase assay on poly (dT) Lanes

1 and 2, control reaction with DNA polymerase a-primase lacking

TAg or vice versa; lanes 3 and 4, 0.2 U and 0.4 U of human; lanes 5

and 6, 0.2 U and 0.4 U of murine DNA polymerase a-primase The

approximate sizes of the reaction products are indicated on the right in

nucleotides (nt).

with SV40 DNA replication Figure 5 shows that murine

DNA polymerase a-primase is indeed incapable of

replica-ting JCV DNA when the replication complex contains

SV40 TAg (columns 2–3) The reciprocal experiment to

investigate whether JCV TAg would relieve the species

specificity of SV40 DNA replication is not feasible as JCV

TAg is incapable of supporting SV40 DNA replication

[32,33]

Discussion

It has been firmly established that the species specificity of

lytic infection by the polyomaviruses SV40 and PyV is

determined at the level of DNA replication both in vivo and

in vitro by the nature of the host DNA polymerase

a-primase complex [2,19,21–27] Here we report that the

closely related JC virus does not show such a strict

specificity in its DNA replication in vitro Although murine

DNA polymerase a-primase is approximately 50% less

efficient than is its human counterpart in the replication of

JCV DNA in our assays (Figs 1–5), nucleotide

incorpor-ation by this complex is significantly above the values

determined in the SV40 DNA replication systems (Figs 1,2

and 4 [21,26,27]) In the purified initiation system of JCV

DNA replication, murine DNA polymerase a-primase can

catalyse primer formation at the JCV origin (Fig 3), a

reaction the murine complex does not support in the SV40

system [22,26,27]

Importantly, we show that nucleotide incorporation in

the JCV system by murine DNA polymerase a-primase is

dependent upon the JCV DNA replication origin (Fig 1B)

and results in DpnI-resistant products (Fig 2), indicating

that it is due to bona fide DNA replication and not a

consequence of filling in of gaps or other short patch repair

events The fact that we observe a complete round of

plasmid replication in murine cell extracts indicates that

other essential replication proteins, such as DNA

poly-merases d and e, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), topoisomerase I and DNA ligase are not responsible for JCV species specificity in vitro Our murine FM3A cell extracts contain relatively low levels

of endogenous RPA and are therefore supplemented with human RPA However, if this is left out we nevertheless observe significant, albeit overall less efficient, incorporation

by both murine and human DNA polymerase a-primase (data not shown) indicating that RPA also is not a species-specific factor

In apparent contradiction of our results, Feigenbaum

et al [8] showed that cultured nonpermissive hamster glial cells were unable to replicate transfected JCV DNA Their observation and our data could be reconciled if the murine but not the hamster cellular DNA replication machinery were permissive for JCV DNA replication We consider this unlikely A more likely reason for the discrepancy between Feigenbaum’s data and our own is the difference in the

state of the DNA in the two assays Transfected DNA will become associated with histones to form chromatin in the cell nucleus whereas our in vitro assays are carried out with naked plasmid DNA Nucleosomes may interfere with the initiation of replication and evidence exists that the binding

of transcription factors to sites in the regulatory regions adjacent to the replication origin of the SV40 chromosome may play a role in relieving such nucleosome repression [48– 52] In vivo JCV DNA replication shows a greater depend-ency on such flanking regions than does SV40 [33] It could

be that JCV species specificity is governed by a host factor such as nuclear factor I (NF-I) required to facilitate initiation of replication in vivo but not in vitro, perhaps by alleviating nucleosome repression or by assisting in origin unwinding under conditions of altered template DNA superhelicity [50] The findings that JCV DNA replication is stimulated by NF-I in vivo but not in vitro support this explanation [50] This view is consistent with the knowledge that introducing an SV40 sequence into the JCV genome

Fig 4 SV40 and JCV DNA replication using the monopolymerase system The SV40 system contains SV40 TAg and DNA with the SV40 replication origin (A); the JCV system con-tains JCV TAg and DNA with the JCV rep-lication origin (B) DNA polymerase (0.25 and 0.5 U) of the indicated DNA polymerase a-primase complexes were added (H4, human, columns 2–3; M4, murine, columns 4–5; –Pol, none, panels (A) and (B), column 1) Enzyme activities were determined beforehand with a DNA polymerase assay on activated calf thymus DNA Standard deviations from three experiments are indicated as error bars The experiments in (A) and (B) were performed in parallel.

can extend the host range of JCV replication in vivo [52].

Alternatively, rodent, but not human, chromatin might

contain a nondiffusable factor that inhibits JCV

TAg-dependent DNA replication or the phosphorylation of JCV

TAg is different in human and mouse cells interfering with

the replication activity in vivo but not with the purified

baculovirus-expressed protein This explanation is

consis-tent with findings that specific residues of SV TAg must be

phosphorylated whereas other may not be [17]

In summary, the available evidence strongly suggests

that, although DNA replication is the species-specific

process common to JCV, SV40 and PyV, different host

factors in each case ultimately determine the restriction of

virus propagation to a particular host For SV40 and PyV

these are, respectively, the p180 and p48 subunits of DNA

polymerase a-primase in initiation of DNA replication

[22–24,26,27,53] For JCV a different level of control

appears to be in operation although we cannot rule out

that the lower initiation efficiency of murine DNA

polymerase a-primase is at least in part involved when

compounded by other factors not present in our assays

formation of an active initiation complex with murine DNA polymerase a-primase (Fig 5), mimicking the molecular basis of SV40 host specificity We recently showed that species specificity of SV40 DNA replication is probably the consequence of a failure of SV40 TAg to undergo a productive functional interaction with C-terminal elements

of the murine p180 subunit of DNA polymerase a-primase [26,53] Our results imply that JCV TAg is not, or is to a much lesser extent, inhibited in these interactions SV40 and JCV TAg share 72% sequence identity with most nonhomology towards the C-termini of the proteins [31] The cell-free in vitro JCV DNA replication system would be useful in determining which regions of SV40 TAg are responsible for its species–specific interactions with host DNA polymerase a, for instance by studying the activity of chimeric TAg polypeptides derived in part from SV40 and

in part from JCV sequences

Acknowledgements

We thank J Fuchs and A Schneider for technical assistance This work was financially supported by the Deutsche Forschungsgemeinschaft (Na190/12 and Na190/13-1) and the EC (CT970125) The IMB is a Gottfried-Wilhelm-Leibniz-Institut and financially supported by the federal government and the Land Thu¨ringen.

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