The ribonucleotide reductase domain of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is essential for R1 antiapoptotic function

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The ribonucleotide reductase domain of theR1 subunit of herpes simplex virus type 2ribonucleotide reductase is essential forR1 antiapoptotic function

Stephane Chabaud,13 A. Marie-Josee Sasseville,1

Seyyed Mehdy Elahi,24 Antoine Caron,2 Florent Dufour,1

Bernard Massie2,3,4 and Yves Langelier1,3,5

Correspondence

Yves Langelier

[email protected]

1Centre de Recherche du Centre Hospitalier de l’Universite de Montreal and Institut du Cancerde Montreal, Hopital Notre-Dame, 1560 Sherbrooke Est, Montreal, QC H2L 4M1, Canada

2Institut de Recherche en Biotechnologie, 6100 ave Royalmount, Montreal, QC H4P 2R2,Canada

3Departement de Microbiologie et Immunologie, Universite de Montreal, QC, Canada

4INRS-IAF, Universite du Quebec, Laval, QC H7N 4Z3, Canada

5Departement de Medecine, Universite de Montreal, QC, Canada

Received 13 July 2006

Accepted 4 October 2006

The R1 subunit (ICP10) of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR),

which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of

about 400 aa, protects cells against apoptosis. As the NH2 domain on its own is not antiapoptotic,

it has been postulated that both domains of R1 or part(s) of them could be necessary for this

function. Here, N- and C-terminal deletions were introduced in HSV-2 R1 to map the domain(s)

involved in its antiapoptotic potential. The results showed that, whereas most of the NH2

domain including part of the recently described putative a-crystallin domain is dispensable for

antiapoptotic activity, it is the integrity of the structured RR domain that is required for protection.

As the a-crystallin domain appears to play an important role in protein folding and oligomerization,

the N-terminal boundary of the antiapoptotic domain could not be defined precisely. In addition,

this study provided evidence that overexpression of HSV-2 R2 at levels up to 30-fold more

than HSV-2 R1 did not decrease protection from tumour necrosis factor alpha, indicating that

the R1 surface where R2 binds is not involved in antiapoptotic activity. Importantly, this result

suggests that the co-expression of both RR subunits during the lytic cycle should not affect

protection from this cytokine.

INTRODUCTION

Herpesviruses of the alpha and gamma subfamilies encodea DNA polymerase, as well as enzymes involved in generat-ing deoxyribonucleotides, notably ribonucleotide reduct-ase (RR), an enzyme consisting of two subunits, R1 and R2.Therefore, they can replicate without the need for thesecellular S-phase enzymes. The alphaherpesviruses herpessimplex virus (HSV) and varicella-zoster virus establish

latency in non-dividing neuronal cells. As these neuroneshave shut down the synthesis of their own RR, the virallyencoded R1 protein, through association with its comple-mentary viral R2 subunit, is essential for reactivation byproviding deoxyribonucleotides for viral DNA replication(Jacobson et al., 1989).

The R1 subunits of human HSV-1 and HSV-2 (also namedICP6 and ICP10) possess, in addition to a C-terminal RRdomain of ~745 aa, an N-terminal domain of ~400 aathat is not found in HSV R1 proteins of species other thanprimates. As the unique NH2 domain is dispensable for RRactivity (Conner et al., 1992; Massie et al., 1998) and HSV-1R1 begins to be expressed earlier in the lytic cycle than its R2partner (Clements et al., 1977; Preston et al., 1978), it hasbeen suggested that the protein could have additionalfunction(s) through its NH2 domain (Conner et al., 1994).

3Present address: LOEX, Hopital du Saint-Sacrement, QC G1S 4L8,Canada.

4Present address: Faculte de Medecine Veterinaire, Saint-Hyacinthe,QC J2S 7C6, Canada.

Details of the construction of plasmids and viruses expressing HSV-2R1 (strain 333) and its deletion mutants are available as supplementarymaterial in JGV Online.

384 0008-2383 G 2007 SGM Printed in Great Britain

Journal of General Virology (2007), 88, 384–394 DOI 10.1099/vir.0.82383-0


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Two additional activities have been associated with HSV-2R1 (Chabaud et al., 2003; Langelier et al., 2002; Perkinset al., 2002a). First, our observation that R1(D2–357), anHSV-2 R1 derivative with its first 357 residues deleted, wasproapoptotic (Massie et al., 1998) led to the finding thatfull-length HSV-2 R1 has an antiapoptotic function thatcan protect cells from apoptosis induced either by expres-sion of R1(D2–357) or by death receptor triggering bytumour necrosis factor alpha (TNF) or Fas ligand (FasL)(Langelier et al., 2002). The demonstration that the NH2

domain on its own was not antiapoptotic ruled out thehypothesis that this new function could be attributablesolely to the ablated domain, and it was postulated thatboth domains of R1 or part(s) of them could be necessaryfor the function (Langelier et al., 2002). Evidence was alsoprovided from infection experiments with two HSV-1 R1deletion mutants (hrR3 and ICP6D) showing that, in addi-tion to its RR function essential for viral reactivation, theHSV-1 R1 could contribute to viral propagation by pre-venting apoptosis induced by TNF (Langelier et al., 2002).Secondly, the presence in HSV R1 proteins of a stretchexhibiting weak similarity to the a-crystallin domain of thesmall heat-shock proteins (sHsps) led to the observationthat HSV-2 R1 displays a chaperone activity as efficient asHsp27. As mammalian R1, which does not exhibit similarityto an a-crystallin domain, does not have either chaperone orantiapoptotic activity, it was proposed that the a-crystallindomain of HSV-2 R1 could play an important role in theantiapoptotic function of the protein (Chabaud et al., 2003).

Genome sequencing of betaherpesviruses including human,chimpanzee, rhesus, mouse, rat and tupaia cytomegalovirus(CMV) as well as human herpesviruses 6 and 7 identified anopen reading frame with homology to R1 but not one for R2.Sequence alignments indicated that essential RR catalyticresidues were missing in these betaherpesvirus R1 proteins,and biochemical characterization of three of these demon-strated that they did not exhibit RR activity (Lembo et al.,2004; Patrone et al., 2003; Sun & Conner, 1999). Randomtransposon mutagenesis of the murine CMV genomeidentified the R1 gene as blocking apoptosis induced bythe virus itself in endothelial cells (Brune et al., 2001).Evidence has also been provided that human CMV R1 cancontribute towards protection from Fas-induced apoptosis(Patrone et al., 2003).

Studies to assess the role of the R1 antiapoptotic functionin HSV-2 pathogenesis, including reactivation from latency,will require an HSV-2 mutant defective in R1 antiapoptoticactivity. However, as RR activity is required for viral DNAreplication during reactivation in animal ganglion neurones,this mutant must be functional in RR activity withoutbeing proapoptotic, as is R1(D2–357). Here, by constructingN- and C-terminal deletions in HSV-2 R1, we mapped thedomain of HSV-2 R1 involved in its antiapoptotic potent-ial. In addition, we have provided evidence that theR2-binding surface is not involved in protection fromTNF-induced apoptosis.

METHODS

Cell lines, plasmids and viruses. Conditions for the culture ofHeLa, A549-tTA and 293-rtTA cells were as described previously(Massie et al., 1998). Details of the construction of plasmids andviruses expressing HSV-2 R1 (strain 333) and its deletion mutantsare available as supplementary material (available in JGV Online).

To isolate A549 cell lines inducibly expressing HSV-2 R1 fused to greenfluorescent protein (R1–GFP), A549-tTA cells were co-transfectedwith pAdTR5-R1–GFP and pTK-Hygro using the polyethyleniminemethod (Durocher et al., 2002) in the presence of anhydrotetracyc-line (15 ng ml21) to repress R1–GFP expression. After hygromycinselection at 200 mg ml21, resistant colonies were pooled. After induc-tion of R1–GFP expression following overnight removal of the an-hydrotetracycline, several single fluorescent cells were isolated with amicromanipulator (Caron et al., 2000). Cell lines were derived bygrowing these cells in the presence of hygromycin (100 mg ml21) andanhydrotetracycline (15 ng ml21).

For R2 co-infection experiments, an HSV-2 R2-expressing virus,AdCR5-R2, was used in combination with a virus expressing a newtransactivator, AdCMV-cTA, derived from the cumate induciblesystem (Mullick et al., 2006). AdCMV-cTA was constructed usingstandard recombination in 293 cells (Massie et al., 1998), whereasAdCR5-R2 containing the HSV-2 R2 gene derived from pSVRR2

HSV-16(Lamarche et al., 1990) was constructed using a positive selectionmethod based on the adenovirus (Ad) protease (Elahi et al., 2002).

Transfection. HeLa cells were seeded 1 day before transfection in60 mm dishes at a concentration of 56105 cells per dish or insix-well plates at 1.56105 cells per well. The calcium phosphatetechnique was used to transfect 25 mg pAdCMV5-R1 or pAdCMV5-R1(D2–249) plasmid ml21, 50 mg pAdCMV5 (empty vector used ascontrol), pAdCMV5-R1(D2–312) or pAdCMV5-R1(D2–357) ml21

or 100 mg pAdCMV5-R1(1–1113) or pAdCMV5-R1(1–1123) ml21.After 24 h, cells were washed twice with sterile PBS and 1 ml (perwell) or 2.5 ml (per dish) of fresh medium was added with anappropriate concentration of Tet system-approved fetal calf serum(TSA-FCS; Clontech).

Infection. A549-tTA cells were plated at a concentration of 36105

cells per well (six-well plate) or 16106 cells per 60 mm dish 1 daybefore the experiment. Cells were infected using conditions toenhance viral adsorption (Langelier et al., 2002). The m.o.i. ofAdTR5-R1 (5 p.f.u. per cell), AdTR5-R1–GFP (25 p.f.u. per cell),AdCMV5-GFP-R1 (50 p.f.u. per cell), AdTR5-GFP (50 p.f.u. percell), AdTR5-R1(D2–357) (50 p.f.u. per cell), AdTR5-R1(D2–398)(250 p.f.u. per cell), AdTR5-R1(D2–496) (250 p.f.u. per cell),AdTR5-R1(1–983) (50 p.f.u. per cell) and AdTR5-R1(1–1081)(100 p.f.u. per cell) were chosen to obtain roughly similar levels ofexpression of the different recombinant proteins.

To evaluate the effect of R2 overexpression on R1 antiapoptoticpotential, A549-tTA cells were co-infected with increasing m.o.i ofAdCR5-R2 and 50 p.f.u. per cell of AdCMV5-cTA. The mediumwas removed 24 h later and the cells were reinfected for 7 h with5 p.f.u. per cell of AdTR5-R1 or mock infected. When used, thepeptidomimetic inhibitor of HSV RR, BILD1633 (BoehringerIngelheim) (Moss et al., 1996), was added 6 h before infection andmaintained throughout the assay.

Apoptosis assays. To determine the antiapoptotic potential of theproteins, apoptosis was induced by adding either cycloheximide(CHX) (15 mg ml21; Sigma) alone as a control or CHX (15 mg ml21)plus human recombinant TNF (2.5 ng ml21; Sigma). Fc : FasL(a recombinant hexameric form of human FasL) (Holler et al.,2003), kindly provided by P. Schneider (University of Lausanne,

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Antiapoptotic domain of the R1 of HSV RR


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Switzerland), was used at 25 ng ml21 with CHX at 15 mg ml21.Generally after 7 h of treatment, the percentage of apoptotic cellswas scored by microscopic observation in at least ten randomlyselected fields, as described previously (Langelier et al., 2002). Cellswere then harvested for caspase-3 activity measurements, as des-cribed previously (Langelier et al., 2002). The percentages of TNF-specific apoptosis and TNF-specific caspase-3 activity were evaluatedusing formulas that eliminated the intrinsic toxicity of some proteinsand CHX as follows: (i) % TNF-specific apoptosis=[(% apoptosisin CHX+TNF-treated cells 2 % apoptosis in CHX-treated cells)/(100 2 % apoptosis in CHX-treated cells)]6100 (Baumler et al.,2003); (ii) TNF-specific caspase-3 activity=caspase-3 activity inCHX+TNF-treated cells 2 caspase-3 activity in CHX-treated cells.To determine the proapoptotic activity of the recombinant proteins,cells were incubated with 1 ml (per well) or 2.5 ml (per dish) offresh medium containing 0.5 % TSA-FCS for 8 h after infection or30 h after transfection.

Immunoblot analysis and protein solubility. Crude total proteinextracts were obtained by sonication in 80 mM Tris/HCl (pH 6.8),2 % SDS and 6 M urea. SDS-PAGE and immunoblotting were per-formed with 10 mg protein per well, as described previously(Lamarche et al., 1996). For HSV-2 R1 detection, the rabbit poly-clonal anti-HSV-2 R1 antiserum 168R1 (Langelier et al., 1998) oranti-HSV R1 mouse monoclonal antibody (mAb) 932 (Ingemarson& Lankinen, 1987) was used. For HSV-2 R2 detection, the polyclo-nal antibody (pAb) P9 was used (Cohen et al., 1986a). A rabbitpolyclonal antibody generated using bacterially expressed GFP wasused for GFP detection. A SuperSignal detection kit (Pierce) wasused according to the manufacturer’s instructions. To determineprotein solubility, cell extracts in HD buffer [50 mM HEPES(pH 8.1), 2 mM DTT] were sonicated and centrifuged at 100 000 gfor 1 h at 4 uC. Cell extracts in RIPA buffer [20 mM Tris/HCl(pH 7.4), 150 mM NaCl, 0.1 % SDS, 1 % NP-40, 1 % sodium deoxy-cholate, 1 mM PMSF] were incubated for 15 min at 4 uC beforesonication, followed by centrifugation at 16 000 g for 30 min at4 uC. The supernatant fraction was removed and stored at 280 uC.The pellet fraction was resuspended in the same buffer as the super-natant and homogenized by sonication before storage. The percen-tage solubility was evaluated by immunoblotting appropriateamounts of both fractions with the antiserum 168R1, as describedpreviously (Langelier et al., 2002).

RR assay. To determine the RR activity of the different R1 pro-teins, infected A549-tTA cells or transfected HeLa cells were used toprepare crude protein extracts, as described previously (Lamarcheet al., 1996). R1 activity was determined by adding excess amountsof HSV-2 R2, partially purified as described previously (specificactivity, 200 U mg21), to limiting amounts of R1 (Lamarche et al.,1996). One unit of RR was defined as the amount of enzyme thatgenerated 1 nmol dCDP min21.

RESULTS

Free N and C termini of full-length R1 are notnecessary for antiapoptotic activity

In a first step towards mapping the R1 domain(s) importantfor its antiapoptotic activity, we examined the importanceof free N and C termini of the full-length HSV-2 R1, aprotein of 1144 aa, by creating GFP chimeras as illustratedin Fig. 1 and by infecting A549-tTA cells with Ad recom-binants expressing the chimeric proteins. When observed byfluorescence microscopy, both chimeras exhibited a cyto-plasmic localization, in sharp contrast to GFP, which was

observed equally in both the cytoplasm and the nucleus(Fig. 2a). The diffuse fluorescence of both chimeras, whichwas similar to the immunofluorescence staining observedfor the native R1, suggested that both proteins were soluble.This was confirmed by analysis of protein solubility afterfractionation by centrifugation in HD buffer (Table 1).

Protection from TNF by recombinants expressing either thechimeras, native R1 or GFP was evaluated by measuringapoptosis by microscopic observation and an in vitrocaspase-3 activity assay (Fig. 2b). The results showed thatboth chimeras were as effective as native R1 in protectingcells, whereas, as previously observed by us (Langelier et al.,2002), GFP was not protective. The chimeras also protectedA549-tTA cells from apoptosis triggered by R1(D2–357)expression (data not shown). The data indicated that freetermini are not required for the R1 antiapoptotic function.Interestingly, the addition of GFP at the C terminuscompletely eliminated RR activity, whereas fusion at the Nterminus did not affect this activity (Fig. 1).

Inducible HSV-2 R1–GFP expression leads toTNF resistance

The observation that the R1–GFP chimeric protein was aseffective as native R1 in protecting cells from TNF-inducedapoptosis led us to establish inducible A549-tTA cell lines,where expression of the chimeric protein was driven byTR5, a tetracycline-responsive promoter (Massie et al.,1998). Six independent single-cell clones were isolated bymicromanipulation of fluorescent cells after briefly turningon chimeric protein expression and thereafter growing themin the presence of anhydrotetracycline. One inducible line,A549-tTA-HSV-R1–GFP, was selected from two undetect-able-background/high-inducer lines for detailed study.

When chimeric protein expression was kept in the OFF statein the presence of anhydrotetracycline, A549-tTA-HSV-R1–GFP cells did not express detectable levels of R1–GFP, asshown by immunoblotting (Fig. 3a), and were destroyedby CHX+TNF treatment as efficiently as their parentalcounterparts. As expected, switching on R1–GFP expres-sion following anhydrotetracycline removal induced TNFresistance with a time course paralleling the detection ofGFP fluorescence by microscopy (Fig. 3b). At the 36 htime point, TNF resistance (>95 %) was equivalent tothe level previously described as being maximal in A549-tTA cells infected with Ad recombinants expressing HSV-2R1 (Langelier et al., 2002). At this time, the R1–GFPconcentration quantified by immunoblotting represented0.1 % of the total cell protein (data not shown). As thisconcentration was equivalent to the minimal level of HSV-2R1 necessary for maximal protection (Langelier et al., 2002),it could be concluded that the addition of GFP at theR1 C terminus did not affect its antiapoptotic potential.Moreover, we observed that, at 36 h after induction of R1–GFP, A549-tTA-HSV-R1–GFP cells were also resistant toFc : FasL-induced apoptosis (data not shown). These resultsshowed that, in addition to Ad recombinant infection and

386 Journal of General Virology 88

S. Chabaud and others


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transient transfection, a third experimental strategy, namelya stably transfected inducible cell line, can be used to studyHSV-2 R1 antiapoptotic potential.

A major part of the HSV-2 R1 NH2 domain isnot essential for protection from TNF-inducedapoptosis

The functional importance of the NH2 domain of HSV-2R1 in the control of cell death was first suggested by thecytotoxicity of R1(D2–357), an HSV-2 R1 subunit with adeletion of its first 357 aa (Massie et al., 1998). Sub-sequently, the observation that the NH2 domain per se wasinsufficient for protection from TNF-induced apoptosissuggested that both domains of R1 or part(s) of them werenecessary for antiapoptotic activity (Langelier et al., 2002).To assess the importance of different segments of theNH2 domain, including the recently described a-crystallin-like domain (Chabaud et al., 2003), a series of deletionmutants was constructed either in CMV5 promoter-driven

expression plasmids or in TR5-inducible Ad recombinantsas illustrated in Fig. 1. The concentration of plasmidstransfected into HeLa cells and the m.o.i of Ad recombinantsused to infect A549-tTA cells were adjusted to obtainroughly equivalent accumulation of recombinant proteins(Fig. 4a). By measuring apoptosis by microscopic observa-tion and an in vitro caspase-3 activity assay, the proapoptoticactivity (Fig. 4b) and protection from TNF (Fig. 4c) of thedifferent truncated proteins were evaluated. The formulasused to calculate the percentages of TNF-specific apoptosisand the TNF-specific caspase-3 activity eliminated the pro-apoptotic activity of CHX and of some truncated proteins.As our previous work had shown that R1(D2–357) wasmostly insoluble (Massie et al., 1998), protein solubility wasassessed by immunoblot analysis after fractionation bycentrifugation in HD or RIPA buffer (Table 1).

The deletion D2–249, which removes a large part of ahydrophilic region located between aa 130 and 294, gavea non-cytotoxic soluble protein with full antiapoptotic

NH2 domain RR domain

HSV-2 R1 (R1)

a-Crystallin domain Helical NH2 domain a/b-barrel domain - Flexible tail

AdTR5-R1_GFPAdCMV5-GFP_R1

N-terminal deletionspAdCMV5-R1(D2_249)pAdCMV5-R1(D2_312)

pAdCMV5-R1(D2_357)AdTR5-R1(D2_398)AdTR5-R1(D2_496)

pAdCMV5-R1(D107_446)pAdCMV5-R1(D378_445)

C-terminal deletions

AdTR5-R1(1_496)AdTR5-R1(1_834)_GFP

AdTR5-R1(1_983)AdTR5-R1(1_1081)

pAdCMV5-R1(1_1113)pAdCMV5-R1(1_1123)

GFP

294400

5841124 Pr

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tion

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ityRR

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ivity

+ _ 62

+ _ <0.5+ _

_ <0.5_

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+ + 55

+ _ 58+ _

ND

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ND

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__ ND

__ ND__ ND

__ ND__ ND_+ ND

GFP

GFP

Fig. 1. Schematic representation and summary of the characterization of truncated HSV-2 R1 proteins. The full-length R1 has1144 residues. The NH2 domain contains an initial region exhibiting a lower similarity between the two HSV types (open box)and an a-crystallin-like domain (filled box) (Chabaud et al., 2003). The RR domain contains two main subdomains termed thehelical NH2 domain (shaded box) and the a/b-barrel domain (hatched box). Protection from TNF and toxicity was determinedas described in Figs 2 and 4. RR specific activity, expressed in nmol min”1 mg”1, was calculated from the percentage ofsoluble R1 in the S100 extract, as described in Methods. ND, Not determined. All of the five C-terminal truncated proteinsshould be RR inactive in vivo, as they are missing essential catalytic residues.




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activity. The deletion D2–312, which removes all of theamino acids preceding the a-crystallin domain, also gavea non-cytotoxic soluble protein, but this protein showeda slight decrease in antiapoptotic activity. Included as anegative control in our analysis of the antiapoptoticpotential, the strongly cytotoxic and barely solubleR1(D2–357) gave an unexpected result in that most of thecells resisting its cytotoxicity were protected from TNF (seephotomicrographs in Fig. 4d). Other deletions where thea-crystallin domain was removed either completely as inR1(D2–398), R1(D2–496) and R1(D107–446) or in part asin R1(D378–445) gave fully insoluble products. None ofthese four truncated proteins was either proapoptotic orprotective against TNF-induced apoptosis [data not shownfor R1(D107–446) and R1(D378–445)]. Taken together, theseresults indicated that nearly all of the NH2 domain includ-ing the first two-thirds of the a-crystallin domain isdispensable for the antiapoptotic function. The data alsosuggested that part of the a-crystallin domain, whichappears to play a role in protein folding, is important forthis function. Finally, the fact that four NH2 deletionmutants showed no mutant-specific phenotype in spite ofthe aggregation of their protein products suggested thatneither the cytotoxicity nor the protection induced by

(a) (b)R1 GFP_R1

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Fig. 2. Free R1 termini are not necessary for antiapoptotic activity. A549-tTA cells were mock-infected (mock) or infected for24 h with AdTR5-GFP (GFP), AdTR5-R1 (R1), AdTR5-R1–GFP (R1–GFP), AdCMV5-GFP–R1 (GFP–R1) as described inMethods. (a) Microscopic observation of R1 immunofluorescence or GFP fluorescence of chimeric proteins or GFP.Immunofluorescence performed with anti-R1 mAb 932 and GFP fluorescence in living cells were photographed usingfluorescence microscopy. (b) Protection from TNF. After 24 h of infection, CHX or CHX+TNF were added and 7 h later,apoptosis was scored by microscopic observation followed by harvesting for caspase-3 activity measurements. Thepercentage of TNF-specific apoptosis (shaded bars) and TNF-specific caspase-3 activity (filled bars; pmol min”1 mg”1) wasevaluated as described in Methods. Values represent the mean±SD of two experiments performed in duplicate. The level ofrecombinant protein expression in total extracts of CHX-treated cells was evaluated by immunoblotting with the anti-R1 pAb168R1 (anti-R1) or the anti-GFP pAb (anti-GFP).

Table 1. Solubility of R1 and mutant R1 proteins

ND, Not determined.

Protein Solubility (%)*

HD buffer RIPA buffer

R1 56 79

R1(D2–249) 44 95

R1(D2–312) ND 75

R1(D2–357) 3 13

R1(D2–398) 0 ND

R1(D2–496) 0 ND

R1(D107–446) 0 0

R1(D378–445) 0 ND

R1(1–496) 35 ND

R1(1–834)–GFP 35 ND

R1(1–983) ND 92

R1(1–1081) ND 68

R1(1–1113) ND 4

R1(1–1123) ND 21

R1–GFP 60 ND

GFP–R1 72 ND

*Values are the means of at least two independent determinations.




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R1(D2–357) appeared to be related to an unspecific cellresponse to the stress created by the accumulation ofinsoluble proteins.

The C-terminal tail is dispensable for protectionfrom TNF

As the NH2 domain alone could not prevent TNF-inducedapoptosis (Langelier et al., 2002), we next attempted tomap which part of the RR domain was important forantiapoptotic activity. Analysis of a mini-library of Adrecombinants expressing randomly truncated R1 proteins(from aa 649 to 834) fused in frame with GFP showed thatnone of these proteins was able to protect against TNF-induced apoptosis (data not shown). We then generatedfour proteins carrying shorter truncations, without fusionto GFP. Only R1(1–1123), the longest protein that included

all of the structured parts of R1, exhibited protection fromTNF (Fig. 5). The ~2-fold reduction in protection ofR1(1–1123) compared with full-length R1 was probablydue to lower solubility of the truncated protein (Table 1).These data demonstrated that the flexible C-terminal tail isdispensable for protection and suggested that C-terminalamino acids beyond the flexible tail are important for thisactivity.

The R2-binding face of HSV-2 R1 is notimportant for antiapoptotic function

As a second approach towards mapping the R1 surface(s)important for antiapoptotic function, we tested the effect ofR1-binding molecules. On binding to R1, R2 covers a largesurface known as the R2-binding face of R1; thus, it couldcompete with binding to R1 of cellular protein(s) control-ling apoptosis. In order to accumulate R2 in large excessover R1, A549-tTA cells were first co-infected with the Adrecombinants AdCR5-R2 (at two different m.o.i.) andAdCMV-cTA for 24 h before being infected for 7 h withAdTR5-R1. Quantification of each protein by comparisonwith purified R1 and R2 standards showed that the R2 : R1ratio was >30 at the higher m.o.i of AdCR5-R2 tested. Thislarge R2 overproduction did not produce any decrease inprotection from TNF (Fig. 6a). HSV-2 R2 expressed alonein parallel controls was not protective (data not shown).This result indicated that the R2-binding surface is notinvolved in R1 antiapoptotic activity. Subsequently, as it hasbeen suggested that the R2 C terminus binds to R1 in acrevice formed between aI and other parts of the protein(Bonneau et al., 1996; Cohen et al., 1986b), we tested theeffect of a peptidomimetic inhibitor of HSV RR that bindswithin this crevice (Liuzzi et al., 1994; Moss et al., 1996).This compound, which exhibits an EC50 of 0.3 mM againstHSV in cell culture (Liuzzi et al., 1994; Moss et al., 1996),added at concentrations up to 10 mM did not alter the R1antiapoptotic activity against TNF (Fig. 6b). This resultsuggested that the R1 surface interacting with the R2 Cterminus is not involved in protection.

DISCUSSION

One of the major findings of the present work was that alarge part of the NH2 domain of HSV-2 R1 (the first 312 aa)could be deleted without significantly affecting protectionagainst TNF and R1(D2–357). The lack of importance of theN terminus of R1 was also supported by results showingthat adding GFP at this extremity altered neither RR activitynor the antiapoptotic function of the protein. This con-clusion was rather unexpected as we had initially thoughtthat the proapoptotic activity of the R1(D2–357) truncatedprotein could be due to antiapoptotic properties of themissing NH2 domain (Langelier et al., 2002). In addition,others had reported evidence that the protection affordedby HSV-2 R1 to 293 cells from staurosporine involvedresidues contained in its first 300 aa (Perkins et al., 2002a, b,2003). It is possible that the domain(s) of R1 implicated

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Fig. 3. Inducible expression of HSV R1–GFP protects cellsfrom TNF-induced apoptosis. Before being seeded in six-wellplates, A549-tTA-HSV-R1–GFP cells were washed twice withmedium containing (OFF) or not (ON) anhydrotetracycline(15 ng ml”1), which represses R1–GFP expression. At the indi-cated times, cells were treated with CHX or CHX+TNF for8 h. (a) The level of recombinant protein expression in totalextracts of CHX-treated cells was evaluated by immunoblottingwith an anti-GFP polyclonal antibody. (b) The percentage ofTNF-specific apoptosis (&, %) was determined under micro-scopic observation 7 h after treatment. The percentage ofGFP-positive cells (X, e) was determined by microscopicobservation of fluorescence in CHX-treated cells. Closed sym-bols, ON state; open symbols, OFF state. Values represent themean±SD of a representative experiment, which was repeatedthree times.




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in the control of apoptosis could differ regarding theproapoptotic stimuli or the cell types used. However, wehave been unable to test this hypothesis directly as ourattempts to demonstrate protection by HSV-2 R1 fromstaurosporine or proapoptotic stimuli other than TNF orFasL and R1(D2–357) were unsuccessful (Langelier et al.,2002). It is noteworthy that Perkins et al. (2002a, 2003) haveascribed the HSV-2 R1 antiapoptotic potential to a puta-tive intrinsic protein kinase activity associated with its NH2

domain, in spite of the fact that extensive biochemicalstudies have demonstrated that both HSV R1 proteins aredevoid of intrinsic protein kinase activity (Conner, 1999;Langelier et al., 1998). The role of the first 312 aa remains to

be determined. However, as these residues are removed byproteolysis in vivo (Ingemarson & Lankinen, 1987) and asthey are not highly conserved between HSV-1 and HSV-2(Nikas et al., 1986), this segment could be of minorimportance. Recent sequencing of three HSVs of Old Worldmonkeys (Perelygina et al., 2003; Tyler & Severini, 2006;Tyler et al., 2005) revealed that the R1 of these virusespossessed a shorter NH2 domain than that of human HSVs,as they lack the poorly conserved segment of aa ~140–310of human HSVs. Interestingly, strong homology of the R1protein of these cercopithecine herpesviruses with R1 ofthe human viruses is observed from aa 319 of HSV-2 R1.Moreover, it is from this position, near the beginning of the

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Fig. 4. Proapoptotic and antiapoptotic acti-vities of N-terminally truncated HSV-2 R1.Cells were transfected with the plasmidpAdCMV5 (Mock), pAdCMV5-R1 (R1),pAdCMV5-R1(D2–249) (249), pAdCMV5-R1(D2–312) (312) or pAdCMV5-R1(D2–357) (357), or mock-infected (Mock), orinfected with the Ad recombinants AdTR5-R1 (R1), AdTR5-R1(D2–357) (357), AdTR5-R1(D2–398) (398) or AdTR5-R1(D2–496)(496) as described in Methods. (a) The levelof recombinant protein expression was eval-uated 30 h after transfection or 8 h afterinfection by immunoblotting with the anti-R1polyclonal antibody 168R1. Quantification ofthe amount of R1 revealed a 5-fold higherlevel in transfected cells than in infectedcells (not shown). (b) Proapoptotic activitywas evaluated 30 h after transfection or 8 hafter infection by scoring the percentage ofapoptosis under microscopic observation inten randomly chosen fields (shaded bars).Cells were then harvested for caspase-3activity measurement (filled bars; pmolmin”1 mg”1). (c) The percentages of TNF-specific apoptosis (shaded bars) and TNF-specific caspase-3 activity (filled bars) wereevaluated 30 h after transfection or 7 h afterinfection as described in Methods. In (b)and (c), values represent the mean±SD oftwo experiments performed in duplicate. (d)Phase-contrast photomicrographs of cellsinfected or not for 12 h and subsequentlytreated for 12 h with CHX or CHX+TNF.




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putative a-crystallin domain, that the two human HSV R1proteins begin to be highly similar.

Our second main observation was that overexpression ofHSV-2 R2 did not decrease HSV-2 R1 protection fromTNF-induced apoptosis. As our previous experiments haveshown that saturation is attained with a 5-fold excess ofR2 over R1 (Lamarche et al., 1994), this result stronglysuggested that the HSV-2 R1 antiapoptotic property doesnot involve its R2-binding surface (~30 % of the totalsurface of R1). The fact that the R2-binding surface maynot be involved in protection from TNF indicates not onlythat other faces of R1 are implicated in this activity but also

that co-expression of both RR subunits during the HSV lyticcycle should not affect R1 antiapoptotic activity. Therefore,as R1 is expressed during a long period of the lytic cycle, itsantiapoptotic activity may play an important role not onlyduring the short period where R1 is expressed alone at thebeginning of the infection but also throughout the lyticcycle.

Results obtained with our other deletion mutants showedthat the reductase domain is involved in the antiapoptoticfunction, but it has been difficult to define precisely theboundaries of the antiapoptotic domain. With the exceptionof R1(D2–312), all of the other deletions on the N-terminal

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Fig. 5. The major part of the RR domain isrequired for protection from TNF. A549-tTAcells were either mock-infected (Mock) orinfected for 24 h at m.o.i described inMethods with the Ad recombinants AdTR5-R1 (R1), AdTR5-R1(1–983) (983), AdTR5-R1(1–1081) (1081) or AdTR5-R1–GFP(R1–GFP), or HeLa cells were transfectedfor 30 h with the plasmids pAdCMV5(Mock), pAdCMV5-R1 (R1), pAdCMV5-R1(1–1113) (1113) or pAdCMV5-R1(1–1123) (1123). Protection from TNF andprotein expression levels were determined7 h after infection or 30 h after transfectionas described in Fig. 4(c). The values repre-sent the mean±SD of two experimentsperformed in duplicate. The level of recom-binant protein expression in total extracts ofCHX-treated cells was evaluated by immuno-blotting with the anti-R1 pAb 168R1.

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side, which removed the putative a-crystallin domain eithercompletely or in part, drastically altered protein solubility.Four of these, R1(D2–398), R1(D2–496), R1(D107–446) andR1(D378–446), were completely insoluble products withoutproapoptotic or antiapoptotic activity. The slightly solubleR1(D2–357) was proapoptotic for a high percentage of cells,but it could also be considered to be antiapoptotic, as cellsresistant to its toxicity were resistant to TNF. From thefact that all of these truncated proteins were lacking theputative a-crystallin domain either totally or in part, itcan be hypothesized that this domain is important forprotein folding and/or oligomerization. Such a role hasbeen demonstrated for the a-crystallin domain of sHsps(reviewed by MacRae, 2000; Studer et al., 2002) andsuggested for the homologous domain present in p23, aco-chaperone of Hsp70 (Garcia-Ranea et al., 2002). Thewild-type RR activity of the small fraction of R1(D2–357)recovered in soluble form in HD buffer, which does notcontain detergent, suggested that this protein could foldproperly, whereas the complete insolubility of R1(D2–398),R1(D2–496), R1(D107–446) and R1(D378–445) indicatedthat these other truncated proteins could be misfolded.As adding 1 M NaCl to the HD buffer used for proteinsolubilization increased the solubility of R1(D2–357) to~50 %, whereas it did not alter the insolubility of R1(D2–398) and R1(D2–496) proteins, it can be further hypothe-sized that the slightly soluble R1(D2–357) forms aggregatesof aberrant oligomers, whereas the completely insolubletruncated proteins form aggregates of misfolded molecules(S. Chabaud & Y. Langelier, unpublished observations). Thedifference in folding of these proteins is the most likelyexplanation for the differences in their anti/proapoptoticpotential. The mechanism underlying the paradoxicalanti/proapoptotic properties of R1(D2–357) is currentlyunknown. One could imagine that, as has been describedfor TNF (reviewed by Gupta, 2002), it could induce botha death process and a survival response. Depending onthe balance between these two signals, cells will engageeither in apoptosis or in survival. The anti/proapoptoticproperties of R1(D2–357) and the fact that the putativea-crystallin domain is absent in cellular R1, which doesnot protect against TNF, suggest that part of this domaincould be important for antiapoptotic activity.

Results obtained with C-terminal deletions demonstratedthat the R1 flexible COOH tail is not necessary for itsantiapoptotic function. Thus, it can be concluded that theC-terminal cysteine pair (Cys1140, Cys1142) involved inthe electron transfer with thioredoxin is not implicatedin this activity. The inactivity of R1(1–1113) was mostprobably due to its very poor solubility. Based on thecrystallographic structure of Escherichia coli R1 and a three-dimensional model of the HSV-1 R1 domain (kindlyprovided by H. Eklund, Swedish University of AgriculturalSciences, Sweden) (Eklund et al., 2001; Uhlin & Eklund,1994) and referring to the numbering of the structuralelements of E. coli R1, the R1(1–1113) ends after the helix aIlocalized on the exterior of the molecule, whereas the active

R1(1–1123) with its additional 10 aa includes a small loopand bJ. As most of these 10 aa are localized inside themolecule in a deep pocket on the R2-binding face, they arenot likely to be involved directly in antiapoptotic activity.Most importantly, as several residues in bJ should interacton one side with aI and on the other side with residuesin aD, bE and notably with the catalytic Cys439, it is likelythat the most important role of bJ in the antiapoptoticfunction is through stabilization of the R1 structure. Ourlonger C-terminal deletions suggested that a region betweenaa 1081 and 1123 could be involved in antiapoptotic acti-vity. Hence, R1(1–1081) and shorter proteins were notaltered in solubility but were all inactive in protection.However, it must be noted once again that these deletionscould affect the overall structure of the protein; hence, site-specific mutagenesis would be required to ascertain theimportance of this region.

Interestingly, a large part of the domain defined here asbeing essential for protection corresponds to the domaincommon to all of the eight known betaherpesviruses R1proteins. Murine and human CMV R1 proteins have beenshown to be implicated in the control of apoptosis (Bruneet al., 2001; Patrone et al., 2003). A multiple sequencealignment of the two HSV R1 proteins with the eightknown betaherpesvirus R1 proteins, which are RR inactive,revealed that the most conserved part (globally >30 %similarity) of the protein is located towards the C ter-minus between HSV-2 R1 aa 834 and 971. Modelling ofthe surface-exposed residues in this segment using theHSV-1 R1 model showed that most of the conservedresidues are located on the face opposite the R2-bindingface (T. Sulea & Y. Langelier, unpublished data). Given itsconservation, the segment aa 834–971 could be importantfor antiapoptotic activity. Importantly, data indicatingthat human CMV R1 could contribute to protection fromFas-induced apoptosis have been obtained with a knock-out viral mutant and not in experiments where the humanCMV R1 was expressed alone. Unfortunately, human CMVR1 when expressed by transfection either in 293 cells(Patrone et al., 2003) or in HeLa cells (S. Chabaud &Y. Langelier, unpublished results) is insoluble. Further site-specific mutagenesis will be required to determine theputative role of these residues in apoptosis protection.

The addition of GFP at the C terminus did not alter theantiapoptotic function but completely eliminated RR acti-vity. The most probable explanation for the effect on thelatter activity is that GFP creates steric hindrance to R2binding. Interestingly, the R1–GFP chimeric protein, withthese attributes, could be an important tool for comple-mentation studies aimed at determining the role of theantiapoptotic function. Moreover, owing to the propertiesof this chimera, we isolated the inducible cell line A549-tTA-HSV-R1–GFP. Upon switching on R1–GFP expression,these cells showed resistance to TNF-induced apoptosis asefficiently as cells transiently expressing R1–GFP either fromtransfection or Ad infection. As they were selected in the




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absence of continuous R1–GFP expression, they representa better model to study the mechanism of action of R1than cells constitutively expressing the protein for severalgenerations (Perkins et al., 2002a) that could have under-gone several modifications through adaptation to R1expression.

ACKNOWLEDGEMENTS

We gratefully acknowledge Claire Guilbault for technical assistance, W.Edward C. Bradley for critical reading of the manuscript, LarsThelander for mAb 932, Boehringer Ingelheim for their generous giftof the peptidomimetic BILD1633 and Hans Ecklund for the HSV-1R1 three-dimensional model. This work was supported by CanadianInstitutes of Health Research grants to Y. L. and B. M. and by a NationalResearch Council of Canada (NRCC) grant to B. M. This is an NRCCpublication no. 47775.

REFERENCES

Baumler, C., Duan, F., Onel, K., Rapaport, B., Jhanwar, S., Offit, K. &Elkon, K. B. (2003). Differential recruitment of caspase 8 to cFlipconfers sensitivity or resistance to Fas-mediated apoptosis in a subsetof familial lymphoma patients. Leuk Res 27, 841–851.

Bonneau, A. M., Kibler, P., White, P., Bousquet, C., Dansereau, N. &Cordingley, M. G. (1996). Resistance of herpes simplex virus type 1to peptidomimetic ribonucleotide reductase inhibitors: selection andcharacterization of mutant isolates. J Virol 70, 787–793.

Brune, W., Menard, C., Heesemann, J. & Koszinowski, U. H. (2001).A ribonucleotide reductase homolog of cytomegalovirus andendothelial cell tropism. Science 291, 303–305.

Caron, A. W., Massie, B. & Mosser, D. D. (2000). Use of amicromanipulator for high-efficiency cloning of cells co-expressingfluorescent proteins. Methods Cell Sci 22, 137–145.

Chabaud, S., Lambert, H., Sasseville, A. M., Lavoie, H., Guilbault, C.,Massie, B., Landry, J. & Langelier, Y. (2003). The R1 subunit ofherpes simplex virus ribonucleotide reductase has chaperone-likeactivity similar to Hsp27. FEBS Lett 545, 213–218.

Clements, J. B., Watson, R. J. & Wilkie, N. M. (1977). Temporalregulation of herpes simplex virus type 1 transcription: location oftranscripts on the viral genome. Cell 12, 275–285.

Cohen, E. A., Gaudreau, P., Brazeau, P. & Langelier, Y. (1986a).Neutralization of herpes simplex virus ribonucleotide reductaseactivity by an oligopeptide-induced antiserum directed againstsubunit H2. J Virol 60, 1130–1133.

Cohen, E. A., Gaudreau, P., Brazeau, P. & Langelier, Y. (1986b).Specific inhibition of herpesvirus ribonucleotide reductase by anonapeptide derived from the carboxy terminus of subunit 2. Nature321, 441–443.

Conner, J. (1999). The unique N terminus of herpes simplex virustype 1 ribonucleotide reductase large subunit is phosphorylated bycasein kinase 2, which may have a homologue in Escherichia coli.J Gen Virol 80, 1471–1476.

Conner, J., Macfarlane, J., Lankinen, H. & Marsden, H. (1992). Theunique N terminus of the herpes simplex virus type 1 large subunit isnot required for ribonucleotide reductase activity. J Gen Virol 73,103–112.

Conner, J., Marsden, H. & Clements, J. B. (1994). Ribonucleotidereductase of herpesviruses. Rev Med Virol 4, 25–34.

Durocher, Y., Perret, S. & Kamen, A. (2002). High-level andhigh-throughput recombinant protein production by transient

transfection of suspension-growing human 293-EBNA1 cells.Nucleic Acids Res 30, E9.

Eklund, H., Uhlin, U., Farnegardh, M., Logan, D. T. & Nordlund, P.(2001). Structure and function of the radical enzyme ribonucleotidereductase. Prog Biophys Mol Biol 77, 177–268.

Elahi, S. M., Oualikene, W., Naghdi, L., O’Connor-McCourt, M. &Massie, B. (2002). Adenovirus-based libraries: efficient generation ofrecombinant adenoviruses by positive selection with the adenovirusprotease. Gene Ther 9, 1238–1246.

Garcia-Ranea, J. A., Mirey, G., Camonis, J. & Valencia, A. (2002).p23 and HSP20/a-crystallin proteins define a conserved sequencedomain present in other eukaryotic protein families. FEBS Lett 529,162–167.

Gupta, S. (2002). A decision between life and death during TNF-a-induced signaling. J Clin Immunol 22, 185–194.

Holler, N., Tardivel, A., Kovacsovics-Bankowski, M., Hertig, S.,Gaide, O., Martinon, F., Tinel, A., Deperthes, D., Calderara, S. &other authors (2003). Two adjacent trimeric Fas ligands are requiredfor Fas signaling and formation of a death-inducing signalingcomplex. Mol Cell Biol 23, 1428–1440.

Ingemarson, R. & Lankinen, H. (1987). The herpes simplex virustype 1 ribonucleotide reductase is a tight complex of the type a2b2composed of 40K and 140K proteins, of which the latter showsmultiple forms due to proteolysis. Virology 156, 417–422.

Jacobson, J. G., Leib, D. A., Goldstein, D. J., Bogard, C. L., Schaffer,P. A., Weller, S. K. & Coen, D. M. (1989). A herpes simplex virusribonucleotide reductase deletion mutant is defective for productiveacute and reactivatable latent infections of mice and for replicationin mouse cells. Virology 173, 276–283.

Lamarche, N., Massie, B., Richer, M., Paradis, H. & Langelier, Y.(1990). High level expression in 293 cells of the herpes simplex virustype 2 ribonucleotide reductase subunit 2 using an adenovirusvector. J Gen Virol 71, 1785–1792.

Lamarche, N., Gaudreau, P., Massie, B. & Langelier, Y. (1994).Affinity of synthetic peptides for the HSV-2 ribonucleotide reductaseR1 subunit measured with an iodinated photoaffinity peptide. AnalBiochem 220, 315–320.

Lamarche, N., Matton, G., Massie, B., Fontecave, M., Atta, M.,Dumas, F., Gaudreau, P. & Langelier, Y. (1996). Production of theR2 subunit of ribonucleotide reductase from herpes simplex viruswith prokaryotic and eukaryotic expression systems: higher activityof R2 produced by eukaryotic cells related to higher iron-bindingcapacity. Biochem J 320, 129–135.

Langelier, Y., Champoux, L., Hamel, M., Guilbault, C., Lamarche, N.,Gaudreau, P. & Massie, B. (1998). The R1 subunit of herpes simplexvirus ribonucleotide reductase is a good substrate for host cellprotein kinases but is not itself a protein kinase. J Biol Chem 273,1435–1443.

Langelier, Y., Bergeron, S., Chabaud, S., Lippens, J., Guilbault, C.,Sasseville, A. M.-J., Denis, S., Mosser, D. D. & Massie, B. (2002).The R1 subunit of herpes simplex virus ribonucleotide reductaseprotects cells against apoptosis at, or upstream of, caspase-8activation. J Gen Virol 83, 2779–2789.

Lembo, D., Donalisio, M., Hofer, A., Cornaglia, M., Brune, W.,Koszinowski, U., Thelander, L. & Landolfo, S. (2004). Theribonucleotide reductase R1 homolog of murine cytomegalovirusis not a functional enzyme subunit but is required for pathogenesis.J Virol 78, 4278–4288.

Liuzzi, M., Deziel, R., Moss, N., Beaulieu, P., Bonneau, A. M.,Bousquet, C., Chafouleas, J. G., Garneau, M., Jaramillo, J. & otherauthors (1994). A potent peptidomimetic inhibitor of HSV ribo-nucleotide reductase with antiviral activity in vivo. Nature 372,695–698.




IP: 54.158.73.222

On: Sat, 13 Aug 2016 07:39:41

MacRae, T. H. (2000). Structure and function of small heat shock/a-

crystallin proteins: established concepts and emerging ideas. Cell Mol

Life Sci 57, 899–913.

Massie, B., Couture, F., Lamoureux, L., Mosser, D. D., Guilbault, C.,Jolicoeur, P., Belanger, F. & Langelier, Y. (1998). Inducible

overexpression of a toxic protein by an adenovirus vector with a

tetracycline-regulatable expression cassette. J Virol 72, 2289–2296.

Moss, N., Beaulieu, P., Duceppe, J. S., Ferland, J. M., Garneau, M.,Gauthier, J., Ghiro, E., Goulet, S., Guse, I. & other authors (1996).Peptidomimetic inhibitors of herpes simplex virus ribonucleotide

reductase with improved in vivo antiviral activity. J Med Chem 39,

4173–4180.

Mullick, A., Xu, Y., Warren, R., Koutroumanis, M., Guilbault, C.,Brousseau, S., Malenfant, F., Bourget, L., Lamoureux, L. & otherauthors (2006). The Cumate gene-switch: a system for regulated

expression in mammalian cells. BMC Biotechnol 6, 43.

Nikas, I., McLauchlan, J., Davison, A. J., Taylor, W. R. & Clements,J. B. (1986). Structural features of ribonucleotide reductase. Proteins

1, 376–384.

Patrone, M., Percivalle, E., Secchi, M., Fiorina, L., Pedrali-Noy, G.,Zoppe, M., Baldanti, F., Hahn, G., Koszinowski, U. H. & otherauthors (2003). The human cytomegalovirus UL45 gene product is a

late, virion-associated protein and influences virus growth at low

multiplicities of infection. J Gen Virol 84, 3359–3370.

Perelygina, L., Zhu, L., Zurkuhlen, H., Mills, R., Borodovsky, M. &Hilliard, J. K. (2003). Complete sequence and comparative analysis of

the genome of herpes B virus (Cercopithecine herpesvirus 1) from a

rhesus monkey. J Virol 77, 6167–6177.

Perkins, D., Pereira, E. F., Gober, M., Yarowsky, P. J. & Aurelian, L.(2002a). The herpes simplex virus type 2 R1 protein kinase (ICP10

PK) blocks apoptosis in hippocampal neurons, involving activationof the MEK/MAPK survival pathway. J Virol 76, 1435–1449.

Perkins, D., Yu, Y., Bambrick, L. L., Yarowsky, P. J. & Aurelian, L.(2002b). Expression of herpes simplex virus type 2 protein ICP10 PKrescues neurons from apoptosis due to serum deprivation or geneticdefects. Exp Neurol 174, 118–122.

Perkins, D., Pereira, E. F. & Aurelian, L. (2003). The herpes simplexvirus type 2 R1 protein kinase (ICP10 PK) functions as a dominantregulator of apoptosis in hippocampal neurons involving activationof the ERK survival pathway and upregulation of the antiapoptoticprotein Bag-1. J Virol 77, 1292–1305.

Preston, V. G., Davison, A. J., Marsden, H. S., Timbury, M. C.,Subak-Sharpe, J. H. & Wilkie, N. M. (1978). Recombinants betweenherpes simplex virus types 1 and 2: analyses of genome structuresand expression of immediate early polypeptides. J Virol 28, 499–517.

Studer, S., Obrist, M., Lentze, N. & Narberhaus, F. (2002). A criticalmotif for oligomerization and chaperone activity of bacterial a-heatshock proteins. Eur J Biochem 269, 3578–3586.

Sun, Y. & Conner, J. (1999). The U28 ORF of human herpesvirus-7does not encode a functional ribonucleotide reductase R1 subunit.J Gen Virol 80, 2713–2718.

Tyler, S. D. & Severini, A. (2006). The complete genome sequence ofherpesvirus papio 2 (Cercopithecine herpesvirus 16) shows evidence ofrecombination events among various progenitor herpesviruses. J Virol80, 1214–1221.

Tyler, S. D., Peters, G. A. & Severini, A. (2005). Complete genomesequence of cercopithecine herpesvirus 2 (SA8) and comparison withother simplexviruses. Virology 331, 429–440.

Uhlin, U. & Eklund, H. (1994). Structure of ribonucleotide reductaseprotein R1. Nature 370, 533–539.



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The ribonucleotide reductase domain of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is essential for R1 antiapoptotic function