genomewide function conservation and phylogeny in the

Genomewide Function Conservationand Phylogeny in the HerpesviridaeM. Mar Alba1, Rhiju Das2, Christine A. Orengo3, and Paul Kellam1,4

1Wohl Virion Centre, Department of Immunology and Molecular Pathology; 2Centre of Mathematics and Physical SciencesApplied to Life Science and Experimental Biology; 3Biomolecular Structure and Modeling Unit, Department of Biochemistry,University College London, London W1T 4JF, UK

The Herpesviridae are a large group of well-characterized double-stranded DNA viruses for which manycomplete genome sequences have been determined. We have extracted protein sequences from all predictedopen reading frames of 19 herpesvirus genomes. Sequence comparison and protein sequence clustering methodshave been used to construct herpesvirus protein homologous families. This resulted in 1692 proteins beingclustered into 243 multiprotein families and 196 singleton proteins. Predicted functions were assigned to eachhomologous family based on genome annotation and published data and each family classified into seven broadfunctional groups. Phylogenetic profiles were constructed for each herpesvirus from the homologous proteinfamilies and used to determine conserved functions and genomewide phylogenetic trees. These trees agreed withmolecular-sequence-derived trees and allowed greater insight into the phylogeny of ungulate and murinegammaherpesviruses.

Viruses contain relatively small genomes and the geneproducts encoded by the genomes are typically in-volved in a restricted number of functions, includingrecognition and entry into cells, specific replication ofthe viral genome, and formation of new virus particles.Some viruses with very small genomes contain <10open reading frames (e.g., retroviruses and papilloma-viruses), whereas others are relatively large and encodefor a few hundred gene products (e.g., poxviruses).Among viruses with large genomes, some of the bestcharacterized are members of the Herpesviridae. Herpes-viruses are double-stranded DNA viruses known to in-fect mammals, fish, and birds. On the basis of differ-ences in the cellular tropism, genome organization,and gene content, herpesviruses have been classifiedinto three subfamilies: the Alphaherpesvirinae, Betaher-pesvirinae, and Gammaherpesvirinae. A large number ofcompletely sequenced genomes are available coveringall three herpesvirus subfamilies (Table 1). A typicalherpesvirus genome consists of ∼70 to 120 ORFs, al-though human cytomegalovirus (HCMV, HHV-5) mayencode over 220 gene products (Cha et al. 1996). Thethree subfamilies are estimated to have arisen 180 to220 million years ago (McGeoch et al. 1995), before themajor mammal radiation, and as such are a diversegroup of viruses. Apart from a number of essential, or

core, genes, contained on seven conserved gene blocks,each genome has a subset of genes characteristic of thesubfamily and a variable number of ORFs, which arespecific to one or a few closely related viruses.

The determination of sequence homology in genesfrom different organisms is key in identifying con-served functions or pathways (Tatusov et al. 1997; An-drade et al. 1999; Pellegrini et al. 1999). Functionallyrelated proteins often share sequence similarity as con-served sequence motifs. Such information has beenused to construct phylogenetic trees based on thenumber of shared genes between different completelysequenced cellular genomes (Fitz-Gibbon and House1999; Snel et al. 1999; Tekaia et al. 1999) and recentlyto build a gene-content herpesvirus phylogeny using13 herpesvirus genomes (Montague and Hutchison2000). We have also used such a whole-genome ap-proach to gain insight into herpesvirus function con-servation and evolution. A larger number of herpesvi-rus genomes (19) have been included in our study andboth gene content and sequence-alignment-derivedphylogenies have been constructed and compared.

Sequence similarities between the ORFs in the cur-rently available complete genomes have been mappedand used to obtain herpesvirus homologous proteinfamilies (HPFs). We have used the phylogenetic distri-bution of these homologous families (phylogeneticprofiles) to determine the level of gene conservationbetween the viruses at different levels of the Herpes-viridae taxonomy. This has enabled the assignment ofhomologous families to known functions and thestudy of how these functions are distributed within the

4 Corresponding author.E-MAIL [email protected]; FAX. 02-07-6799555.Article and publication are at www.genome.org/cgi/doi/10.1101/gr.149801.

Letter

11:43–54 ©2001 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/01 $5.00; www.genome.org Genome Research 43www.genome.org

herpesviruses. The phylogenetic profiles have beenused to construct phylogenetic trees based on con-served gene function, reflecting the gain and loss offunctions that underlay herpesvirus taxonomy.

RESULTS

Identification of Homologous Protein Familiesand Function AssignmentSequence homology among all proteins derived fromcomplete herpesvirus genomes (Table 1) was deter-mined and used as a basis to construct HPFs (Fig. 1).We identified 243 homologous families that containedtwo or more proteins, comprising 1496 proteins out ofa total of 1692 predicted ORFs in the 19 genomes stud-ied. We observed that ∼80% of the total herpesvirusproteins had homologs in a different herpesvirus,whereas 20% appeared to be unique to particular ge-nomes, sometimes existing as multiple copies (para-logs). Three-dimensional structural information for asubset of herpesvirus proteins validated the homolo-gous family groups. It was not possible to collapse thehomologous families into smaller groups based onsuch structural information. We used GenBank header

files to manually assign functions to the differentHPFs, including those with only one protein member.Functions consisted of both a short definition, such asDNA polymerase, and a broad functional class, for ex-

Figure 1 Schematic representation of a homologous proteinfamily (HPF). Identically shaded boxes represent identified re-gions of each protein that have sequence homology. HPFs areconstructed computationally by identifying one (or more) re-gion(s) of sequence homology (i.e., unfilled oval) that builds thelargest group of sequences. The HPF-conserved sequence regioncan be found in all proteins in the HPF.

Table 1. Herpesvirus Genomes Used to Construct Homologous Protein Families

Subfamily andsublineage Virus name (strain) Acronyms

GenBank Accessionno. ORFs*

Length(Kb)

Alphaherpesviruses�1 Human herpesvirus 1 (17) HSV-11/HHV-1 X14112 77 152�1 Human herpesvirus 2 (HG52) HSV-22/HHV-2 Z86099 77 154�2 Human herpesvirus 3 VZV3/HHV-3 X04370 71 124�2 Equine herpesvirus 1 (Ab4p) EHV-1 M86664 80 150�2 Equine herpesvirus 4 (NS80567) EHV-4 AF030027 79 145�2 Bovine herpesvirus 1 (K22) BHV-1 AJ004801 73 135�3 Gallid herpesvirus 2 (HPR524) GHV-2 AB024414 65 110Betaherpesviruses�1 Human herpesvirus 5 (AD169) HHV-5/HCMV4 X17403 203 229�2 Human herpesvirus 6 A (U1102) HHV-6 (A) X83413 121 159�2 Human herpesvirus 6 B (HST) HHV-6 (B/HST) AB021506 115 161�2 Human herpesvirus 7 (JI) HHV-7 (JI) U43400 107 144Gammaherpesviruses�1 Human herpesvirus 4 (B95-8) HHV-4/EBV5 V01555 86 172�2 Alcelaphine herpesvirus 1 (C500) AHV-1 AF005370 70 130�2 Ateline herpesvirus 3 (73) HVA-36 AF083424 71 108�2 Macaca mulatta rhadinovirus (17577) RRV7 AF083501 80 133�2 Saimiriine herpesvirus 2 HVS8 X64346 76 112�2 Equine herpesvirus 2 (86/87) EHV-2 U20824 79 184�2 Human herpesvirus 8 HHV-8/KSHV9 U75698 82 137�2 Murine herpesvirus 68 (WUMS) MHV-68 U97553 80 119

*Number of open reading frames as extracted from GenBank entry (Benson et al. 1999).1HSV-1; herpes simplex virus-12HSV-2; herpes simplex virus-23VZV; Varicella Zoster virus4HCMV; human cytomegalovirus5EBV; Epstein-Barr Virus6HVA-3; herpesvirus ateles-37RRV; rhesus rhadinovirus8HVS; herpesvirus saimiri9KSHV; Kaposi’s sarcoma associated herpesvirus

Alba et al.

44 Genome Researchwww.genome.org

ample, replication. Uncharacterized proteins were as-signed to the unknown class.

All HPFs that belong to different functional classescan be retrieved from http://www.biochem.ucl.ac.uk/bsm/virus database. In addition, the HPFs can besearched using virus name, functional annotation,keywords, or GenBank protein entry numbers. EachHPF has been assigned a distinct family number (HPF1, HPF 2, etc.).

Phylogenetic Distribution of ProteinHomologous FamiliesWe used the homologous families to build proteinphylogenetic profiles (Pellegrini et al. 1999), in whichfor each homologous family the presence or absence inevery genome was recorded in the form of a binarymatrix, where 1 means presence of at least one proteinfrom the genome and 0 means no protein. In thistype of analysis, paralogous proteins, resulting frommultiple copies of a gene in the same genome, willonly be counted once. The profiles were used todetermine the number of gene functions conserved inpairs of genomes and to construct phylogenetictrees. In addition, the profiles were used to determinethe minimum number of functions conserved at thesubfamily/lineage level and to study the degree ofconservation with respect to the functional class ofthe gene.

The distribution of the number of shared func-tions, based on sequence homology, between any twogenomes across the different Herpesviridae was in accor-dance with the main evolutionary herpesvirus lineages(subfamilies) and sublineages (individual viruses). Arelatively high number of homologous families wereconserved within subfamilies and a much lowernumber conserved between members of differentsubfamilies (Table 2, lower triangle). Using our se-quence-comparison algorithm, the minimum numberof shared homologous families was 26 conserved be-tween the Alpha- and Betaherpesvirinae, and the maxi-mum, 96, was found between the closely relatedHHV6-A and HHV6-B. The number of shared homolo-gous families was more variable within subfamiliesthan between members of different subfamilies. Forexample, the number of shared homologous familieswithin any two Alphaherpesvirinae viruses rangedfrom 52 to 77, but between members of this subfamilyand the other two subfamilies, the range of conservedhomologous families was much narrower, between 26and 30. We also calculated the percentage of homolo-gous families conserved between any two genomes,taken relative to the genome with a smaller number ofdifferent families (Table 2, upper triangle). At least one-third of the homologous families were conserved be-tween any two genomes with respect to the smallestgenome of the pair. Within subfamilies the percentage

varied between 54% (HHV-5 vs. HHV-6) and 100%(HSV-1 vs. HSV-2).

Conservation of Function Withinand Across SubfamiliesWe next focused our attention at the number of ho-mologous families, which formed the core set of pro-teins in the different herpesvirus subfamilies. We de-tected 26 different ORFs that were conserved across theHerpesviridae (Table 3), which is close to previous esti-mations of the minimal herpesvirus genome on thebasis of clear sequence homology (Hannenhalli et al.1995; McGeoch and Davison 1999a). Each of theseORFs formed a separate homologous family, except forthe major and the minor capsid proteins that share aregion of ∼66 amino acids and, therefore, are part ofthe same homologous family. Apart from this commonset of genes, other ORFs were conserved in two sub-families but were absent in the third. In particular, wefound three homologous families that were specific forAlpha- and Gammaherpesviruses and 10 specific forBeta- and Gammaherpesviruses. We did not identifyany homologous families present in all members of theAlpha- and Betaherpesviruses but not present in theGammaherpesviruses. According to this, the Gammaand Beta lineages clearly share more genes with detect-able sequence homology than either of the two withthe Alphaherpesviruses. By computing the ORFs thatwere only conserved in all members of one subfamilybut in no other herpesvirus, we determined the sub-family-specific homologous families. There were 22such homologs for the Alphaherpesviruses, 23 for theBetaherpesviruses, and only 8 for the Gammaherpesvi-ruses. By adding the homologous families conserved atthe level of two or three subfamilies, we obtained 51families totally conserved for Alphaherpesviruses, 59for Betaherpesviruses, and 46 for Gammaherpes-viruses.

Analysis of Different Functional ClassesThe number of homologous families with known func-tion identified in viruses from different lineages wasvariable. The Alphaherpesviruses were the best charac-terized, with between 60% and 80% of the proteins ofany virus having an assigned function. This percentagewas between 55% and 70% for the Gammaherpesvi-ruses. The Betaherpesviruses contained the largestnumber of uncharacterized proteins among the differ-ent lineages. Only about half of the predicted HHV-6and HHV-7 ORFs and about one-fourth of the HHV-5(human cytomegalovirus, HCMV) ORFs have a docu-mented function.

Next we compared the degree of conservation ofthe different homologous families across the wholeHerpesviridae. To do this, we analyzed separately the

Herpesvirus Phylogenetic Profiles

Genome Research 45www.genome.org

Tab

le2.

Num

ber

of

Ho

mo

log

ous

Fam

ilies

Shar

edb

etw

een

An

yTw

oG

eno

mes

HSV

-1H

SV-2

VZ

VEH

V-1

EHV

-4B

HV

-1G

HV

-2H

HV

-5H

HV

-6A

HH

V-6

BH

HV

-7H

HV

-4A

HV

-1H

VA

-3R

RV

HV

SEH

V-2

HH

V-8

MH

V-6

8

HSV

-174

100

8682

8282

8035

3636

3638

4140

3838

3938

38H

SV-2

7475

8682

8283

8035

3636

3638

4140

3837

3937

37VZ

V58

5867

9494

8981

3940

4040

4243

4343

4345

4342

EHV-

161

6263

7710

092

8334

3535

3539

4140

3837

3936

36EH

V-4

6162

6377

7792

8334

3535

3539

4140

3837

3936

36BH

V-1

5960

6066

6672

8136

3737

3739

4140

3939

4039

39G

HV-

252

5253

5454

5365

4042

4242

4345

4343

4345

4343

HH

V-5

2626

2626

2626

2616

754

5458

4551

5149

4748

4446

HH

V-6A

2727

2727

2727

2756

104

9285

4551

5149

4748

4446

HH

V-6B

2727

2727

2727

2756

9610

483

4350

5047

4647

4345

HH

V-7

2727

2727

2727

2756

8281

9645

5151

4947

4844

46H

HV-

428

2828

2929

2828

3333

3233

7471

6968

6768

6863

AH

V-1

2929

2929

2929

2936

3636

3650

7080

8081

8080

76H

VA-3

2828

2928

2828

2836

3636

3648

5670

9097

8090

83RR

V28

2829

2828

2828

3636

3636

5056

6374

8977

9681

HVS

2828

2928

2828

2836

3636

3650

5768

6676

7687

79EH

V-2

2929

3029

2929

2936

3636

3650

5656

5757

7576

71H

HV-

828

2829

2828

2828

3636

3636

5056

6371

6657

8178

MH

V-68

2828

2828

2828

2836

3636

3647

5358

6060

5361

78

Abs

olut

enu

mbe

r(lo

wer

tria

ngle

and

diag

onal

,sh

aded

)or

per

cent

age

with

resp

ect

toth

ege

nom

ew

ithth

elo

wes

tnu

mbe

rof

hom

olog

ous

func

tions

(up

per

tria

ngle

)

Alba et al.


phylogenetic profiles of homologous families that be-longed to different functional classes. The analysis isshown for the structural class (Fig. 2). The size distri-bution of the homologous family, taken as the numberof different viruses represented, was markedly differentfor the seven functional classes (Fig. 3). Genes involvedin nucleotide metabolism and DNA repair were themost conserved, with most of them being in largegroups containing viruses from two or three subfami-lies. Structural proteins, including capsid and tegu-ment proteins, were also well conserved, as were pro-teins from the replication functional class. However,glycoproteins showed a much lower conservation andmost of them belonged to families with a size of 1–3viruses, clearly below the size of a herpesvirus subfam-ily. Proteins identified as being involved in transcrip-tion, as well as proteins in the others group, whichincluded genes involved in virus-host interactions,were also poorly conserved. Finally, the majority ofhomologous families with an as-yet-unknown func-

tion (unknown class) fell into the 1–3 viruses sizerange.

Interestingly, the three proteins that have beenconserved in all Alpha- and Gammaherpesvirusesbut not in Betaherpesviruses belonged to the samefunctional group, nucleotide metabolism/DNA re-pair, namely ribonucleotide reductase small subunit,dUTPase, and thymidine kinase (HPF 28, 29, and 31,respectively). In contrast, homologous families that areexclusively conserved between the Beta- and Gamma-herpesviruses were structural or of unknown function.One HPF, 9, contained the DNA origin-binding proteinfrom the Alphaherpesviruses and the Betaherpesvi-ruses HHV-6/HHV-7. However, this protein showed nohomology to any Gammaherpesvirus protein or to pro-teins from the Betaherpesvirus HHV-5 (human cyto-megalovirus). Homologous families that appeared tobe exclusive to particular herpesvirus subfamilies oc-curred across different functional classes, although 12homologous families corresponded to structural pro-

Table 3. List of Herpesviridae Open Reading Frames with Clear Sequence Conservation in the19 Genomes

Geneblock1

Length ofconservedsequence2

GenBanknumber(HSV-1)

Genename

(HSV-1) Function3 Functional class4

A 750 gi:59530 UL30 DNA polymerase RepA 86 gi:59531 UL31 unknown UnkA 436 gi:59533 UL32 virion protein StrA 42 gi:59536 UL36 tegument protein StrA 285 gi:59539 UL39 ribonucleotide reductase large subunit NucB 667 gi:59527 UL27 glycoprotein B GlyB 599 gi:59528 UL28 transport protein StrB 960 gi:59529 UL29 ssDNA binding protein RepC 714 gi:59552 UL52 helicase-primase complex RepC 39 gi:59554 UL54 immediate-early transactivator TrfD 63 gi:59522 UL22 glycoprotein H GlyD 111 gi:59523 UL24 fusion protein StrD 131 gi:59525 UL25 tegument protein StrD 185 gi:59526 UL26 capsid protease StrE 102 gi:59518 UL18 capsid protein StrE 66 gi:59519 UL19 major capsid protein StrF 767 gi:59507 UL5 helicase-primase complex RepF 66 gi:59506 UL6 minor capsid protein StrF 67 gi:59508 UL7 unknown UnkF 283 gi:59510 UL10 glycoprotein M GlyF 276 gi:59513 UL12 deoxyribonuclease NucF 140 gi:59514 UL13 protein kinases OthF 315 gi:59501 UL152 DNA packaging StrF 143 gi:59501 UL151 DNA packaging StrF 90 gi:59516 UL16 virion protein StrG 218 gi:59503 UL2 uracil-DNA glycosylase Nuc

1Gene blocks are regions where the order of genes is conserved of which seven are present in all Herpesviridaegenomes (A-G).2Conserved sequence regions where sequence homology was clearly detected. These corresponded to a singlecontigous sequence motif in all cases except for DNA polymerase, in which three different motifs wereconserved.3Function as derived from GenBank annotations.4Functional classes: Rep (replication), Nuc (nucleotide metabolism and DNA repair), Str (structural), Trf(transcription), Gly (glycoprotein), Oth (other), Unk (unknown).



teins in Alphaherpesviruses and 12 to genes of un-known function in Betaherpesviruses.

Phylogenetic Reconstruction Basedon Function ConservationPhylogenetic profiles were used to construct phyloge-netic trees based on whole-genome homologous fam-ily content. In this type of tree, the distances betweenthe different viruses are based on the degree of conser-vation of gene functions. Therefore, the topology ofthe tree will be affected by gene loss, gene capture(typically from the host genome in herpesviruses), andextensive sequence divergence beyond the recognitionby the sequence comparison methods used here. Thephylogenetic profiles were bootstrapped 100 times be-fore constructing the trees. To build neighbor-joiningtrees, we explored the use of two types of intergenomicdistance, the fraction of nonshared functions, and thefraction of dissimilar functions (Fig. 4A,B, respec-tively). The branching order of the two trees was thesame for the two approaches and the main differenceswere in the branch lengths. As expected, the distancemethod that used the total of dissimilar functions,

which was not standardized tothe size of the smaller genome,reflected the difference in thenumber of genes per genomemuch better (Fig. 4B). For ex-ample, the branch length forHHV-5, which has approxi-mately twice as many genesthan any other herpesvirus ge-n o m e , w a s l o n g e r t h a nbranches in other parts of thetree. Bootstrap supports, in gen-eral, were very high, with theexception of the split of the twoungulate herpesviruses, alcela-phine herpesvirus 1 (AHV-1)and equine herpesvirus 2 (EHV-2), with bootstrap values of44% and 37%, respectively. Inaddition to neighbor-joiningtrees, we built up a maximumparsimony tree from the sameset of data (Fig. 4C). Again thebranching pattern was thesame, except for the indepen-dent split of AHV-1 and EHV-2as sisters, although, again, thebootstrap value was relativelylow (64%).

The trees based on the phy-logenetic profile clearly re-solved the splits between her-pesvirus subfamilies and sublin-

eages (Table 1). In addition, our data regarding thenumber of shared functions between different sub-families supported previous observations of an earlysplit of the Beta- and Gammaherpesviruses from theAlphaherpesviruses. This branching pattern was ob-served when we simulated a root by using an artificialoutgroup genome that had none of the homologousproteins, that is, a row of 0s in the phylogenetic profile.To compare these trees with a sequence-comparison-based tree, we constructed an alignment of all con-served domains in the 26 ORFs identified as clear ho-mologs in all herpesviruses. These genes have been pre-served throughout herpesvirus evolution and arepresent in one copy per genome. The alignment con-tained 8900 positions and, using 100 bootstrapped datasets, neighbor-joining, UPGMA, and maximum parsi-mony trees were constructed. All trees showed the sametopology and the neighbor-joining tree is shown in Fig-ure 4D. The trees were representative and agreed withprevious phylogenetic trees produced using a smaller setof highly conserved herpesvirus proteins (McGeoch andDavison 1999a).

There was complete consistency between the trees

Figure 2 Phylogenetic profile of homologous proteins families (HPF) known to be involved instructural functions (capsid, tegument, virus assembly). The presence of the family in any ge-nome is indicated by 1 and the absence by 0. Alpha, Alphaherpesviruses; Beta, Betaherpesvi-ruses; and Gamma, Gammaherpesviruses. The HPF numbers are indicated and relate directly toaccompanying data available at http://www.biochem.ucl.ac.uk/bsm/virus_database. HPF 1* isindicated twice because it represents a shared domain present in both the major and minorcapsid proteins of all herpesviruses.

Alba et al.


based on either function conservation or on sequencealignment, for the Alpha- and Betaherpesviruses, withall trees producing the same branching pattern.Among the Gammaherpesviruses, branch differencesoccurred in the positions of the ungulate herpesviruses(AHV-1 and EHV-2) and the murine herpesvirus MHV-68 when comparing the various trees. The position ofthe MHV-8, previously unresolved (McGeoch andDavison 1999b), appeared basal to the rhadinoviruses(all Gammaherpesviruses except HHV-4 in this study)in the alignment-based tree with a bootstrap value of99%. Instead MHV-68 clustered together with the hu-man and primate viruses in the other trees (bootstrapvalues of 87% and 69%). AHV-1 and EHV-2 formed acluster in the neighbor-joining trees based on homolo-gous family conservation. This association is in accor-dance with the hypothesis that herpesviruses have co-evolved with their hosts (McGeoch and Cook 1994;

McGeoch and Davison 1999b). However, the bootstrapvalues were low and the cluster was not observed in theother two trees. Therefore, the result is suggestive butrequires further investigation.

DISCUSSIONThe evolution of herpesviruses has been studied by se-quence-comparison methods using a subset of con-served proteins (McGeoch and Cook 1994; McGeoch etal. 1995; McGeoch and Davison 1999a), by genomecompositional properties such as dinucleotide fre-quency and CG content (Karlin et al. 1994), and byrearrangements of conserved gene blocks within thedifferent genomes (Hannenhalli et al. 1995). Thisstudy of the molecular functions shared in 19 completegenomes in the form of phylogenetic profiles from her-pesvirus HPFs has provided additional information onthe degree of gene conservation at different levels ofthe Herpesviridae taxonomy. The complete genome ap-proach has been successfully used to construct a phy-logenetic tree that, although being in agreement withalignment-derived trees with respect to the best-supported branching events, provides additional in-sights into Gammaherpesvirus evolution.

The rate of gene turnover in herpesviruses appearsto be quite high outside the core of conserved genes.This is reflected in a high number of genes that areunique to a particular herpesvirus and do not havecounterparts in other herpesviruses. This group repre-sents ∼20% of the total herpesvirus ORFs. The majorityof these genes are of unknown function, although itseems likely that many of them were captured from thehost genome during a relatively recent time. Virus-specific genes, including some multigene families, arenot distributed evenly across the Herpesviridae but areparticularly abundant in some subfamilies or viruses.For example, within the Betaherpesviruses, ∼70% ofthe HHV-5 genes appear to be virus specific. A similarfeature is seen for the Gammaherpesvirus MHV-68, forwhich ∼20% of the genes have no sequence homologsin any other herpesvirus.

According to the sequence comparison algorithmused, the Herpesviridae share a set of 26 different ORFsand, therefore, about one-third of their functions arecommon (except for the large HHV-5 genome). Thesecommon functions include replication and nucleotidemetabolism proteins, some structural proteins and gly-coproteins, and a virus gene expression regulatory fac-tor, designated UL54 in HSV-1. The less-well-conservedfunctional groups belong to the transcription, glyco-proteins, and proteins classified as others. These obser-vations, applied to the whole of the herpesvirus family,confirm similar conclusions as those derived from aprotein functional analysis of the well-characterizedherpes simplex virus 1 and its relatives in other hostspecies (McGeoch and Davison 1999a). Within sub-

Figure 3 Distribution of functional classes of homologous fami-lies across the 19 herpesviruses considered. The number of her-pesvirus genomes that contain the functional class are shown onthe X-axis (lowest graph) and the number of homologous familiesin the functional class are shown on each Y-axis.



families, the conservation of function is always >50%,establishing a clear demarcation between subfamilies.Functions that are selectively conserved or eliminated

in certain subfamilies are clearly visible, for example,the conservation of certain enzymes involved innucleotide metabolism in the Alpha- and Gammaher-

Figure 4 Phylogenetic trees based on protein family phylogenetic profiles (A,B,C) or on sequencecomparison of herpesvirus-conserved domains (D). The initial data or alignments were bootstrapped100 times. Neighbor-joining trees were constructed using the fraction of nonshared homologousfamilies (A) or the fraction of dissimilar homologous families (B), as described in the text. Maximumparsimony cladogram built from the phylogenetic profiles (C). Neighbor-joining tree constructed usingconserved regions in 26 herpesvirus open reading frames (D). �, Alphaherpesviruses; �, Betaherpesvi-ruses; and �, Gammaherpesviruses. The rhadinovirus subgroup of the Gammaherpesviruses consists ofthe viruses MHV-68, AHV-1, HVA-3, HVS, RRV, HHV-8, and EHV-2.

Alba et al.


pesviruses but not in the Betaherpesviruses. This hasbeen previously interpreted as the Betaherpesvirus sub-family having abandoned the strategy of supplying en-zymes of nucleotide synthesis for the replication oftheir genomes (McGeoch and Davison 1999a). Fromthis study, we found that the Beta- and Gammaherpes-viruses share more functions than either of these sub-families do with the Alphaherpesviruses. Althoughmany of these proteins are as yet uncharacterized, itseems likely that some will have a virus-structure func-tional role. This is supported by the fact that Alpha-specific genes are mostly from the structural class and,therefore, may be distant relatives of the Beta- andGamma-specific genes. This level of relationship maybe undetectable at the amino acid sequence level butmay become apparent by secondary and three-dimensional structure prediction methods.

Taking into account the estimates for herpesvirusdivergence (McGeoch et al. 1995) and the differencesin the number of shared functions in the different her-pesvirus genomes, we have calculated that, on average,a decrease of ∼7% in shared functions corresponds to20 Myrs. From this we could extrapolate a rate of de-crease of shared gene fraction between two herpesvirusgenomes of about 3.5 � 10�3/Myr. In reality, this is anestimate of the minimum gene turnover, as recentgene duplications, represented as several proteins inthe same homologous family from the same genome,would not enter into this equation. The rate of de-crease of shared gene fraction between prokaryotic ge-nomes can be estimated to be about 1 � 10�4 to3 � 10�4/Myr from prokaryotic genome comparisondata (Snel et al. 1999). Therefore, the gene turnover inherpesvirus genomes is an order of magnitude higherthan in prokaryotic genomes. Similarly, amino acidmutation rates in herpesvirus proteins have been esti-mated to be higher (∼10–100 times) than in corre-sponding proteins in the host genomes (McGeoch andCook 1994).

The construction of phylogenetic trees from genecontent is a relatively new method of phylogenetic in-ference (Fitz-Gibbon and House 1999; Snel et al. 1999;Teichmann and Mitchison 1999; Tekaia et al. 1999)that we have applied to the study of viral genomes.Classical molecular methods, based on the alignmentof individual gene sequences, are subject to the factthat different genes may have different evolutionaryhistories and undergo different types of selective pres-sure. As a consequence, the trees derived from suchgenes or proteins often differ. Instead, phylogenetictrees derived from gene content or molecular functionconservation capture a broader picture and may ac-commodate some of the gene-specific biases. However,phylogeny based on gene content are affected by hori-zontal gene transfer and by differences in the numberof genes in the genomes. Despite these potential prob-

lems, we have successfully applied homologous-familyconservation-based methods to reconstruct a phylog-eny of the Herpesviridae. The tree-branching pattern isin excellent agreement with phylogenies derived fromalignments of conserved amino acid regions.

Differences exist at the level of the murine andungulate rhadinoviruses. The position of MHV-68could not previously be resolved by sequence-comparison-based methods (McGeoch and Davison1999b). MHV-68 appears basal to the rhadinovirusclade in our alignment-based tree, representing thegeneral trend of sequence divergence in the conserveddomains for this virus. However, MHV-68 clusters witha relatively high confidence with primate Gammaher-pesviruses in the three different trees based on ho-mologous family conservation. In addition, a commonsplit for the two ungulate Gammaherpesviruses(AHV-1 and EHV-2) is suggested by using the distance-based methods with phylogenetic profile data. This lat-ter split would be expected by the hypothesis of coevo-lution of herpesviruses with their hosts (McGeoch andDavison 1999b) but is not detectable from sequence-comparison-based methods. Analysis of the homolo-gous families within rhadinoviruses provides furtherinsight into the evolution of this clade. The cluster ofthe murine and primate viruses is supported by twodifferent genes present in these viruses but absent fromthe rest of herpesviruses, namely the viral-cyclin D ho-molog and the latent nuclear antigen (HPF 110 andHPF 111, respectively). These genes are involved in la-tency or interactions with the host and have corre-sponding locations within the different genomes. Inaddition, there are no genes exclusive to the ungulateand murine herpesviruses or to the ungulate and pri-mate rhadinoviruses. However, two homologous fami-lies (HPF 81 and HPF 89, structural and glycoproteingroups, respectively) are present in all Gammaherpes-viruses (including HHV-4/EBV) but absent from MHV-68, possibly reflecting specific gene losses in MHV-68.

The evidence for a common branch for AHV-1 andEHV-2 is not strongly supported by high bootstrap val-ues for the number of shared genes, but specific genesdo give support for the tree topology. A homologousfamily of a putative transmembrane protein (HPF 232)is only present in AHV-1 and EHV-2 and, therefore,could have been present in a common ancestor ofthese two viruses. Also in support of an early branchingof the ungulate viruses is the existence of one gene ofunknown function present in EBV (ORF BZLF2), AHV-1,and EHV-2 but absent from the rest of the rhadinoviruses(HPF 153). Furthermore, a homologous family includingORF BRRF1 from EBV (HPF 97) is present in all rhadino-viruses except the two ungulate viruses. The first twogenes, therefore, could have been lost in a branch com-mon to murine and primate herpesviruses, whereas thelatter could have been lost in the ungulate branch.



Trees based simply on sequence alignment maynot be able to successfully reconstruct distant branch-ing events, especially if the proteins have divergedquickly. Rates of mutation are not uniform betweendifferent organisms and, in the case of pathogens, in-fection of new hosts may lead to accelerated sequencechange in some or all proteins. The basal position ofMHV-68 in the alignment-based tree could be due toan early ancestry of this virus within the rhadinovi-ruses or alternatively to a high rate of amino acid se-quence divergence. If MHV-68 is truly basal to therhadinoviruses, the proximity to the primate Gamma-herpesviruses in the trees based on shared genes wouldimply that MHV-68 and primate viruses have been un-der similar selection pressures for the conservation andloss of gene sets, distinct from those conserved or lostin the ungulate Gammaherpesvirus. An alternativeway to explain the differences between the two typesof trees is that the murine and primate Gammaherpes-viruses are evolutionarily closer, as supported by genecontent trees, but that a high rate of amino acidchange in MHV-68 results in an underestimation oftheir relationship in the alignment-based tree. Forlarge genome viruses, trees based on homologous fam-ily conservation may capture other phylogenetic sig-natures, such as gene loss and acquisition that al-though prone to the errors associated with horizontalgene transfer and secondary losses, may provide higherresolution in cases such as the ones discussed.

Two additional cytomegalovirus genome se-quences, murine cytomegalovirus 1 and rat cytomega-lovirus, were not included in this study. The genome ofmurine cytomegalovirus was sequenced in 1996 (Raw-linson et al. 1996), but, unfortunately, the translatedprotein sequences are not available. The sequence ofrat cytomegalovirus genome (Vink et al. 2000) ap-peared at a late stage of the revision of this paper. Thesetwo viruses belong to the Betaherpesvirus subfamilyand have been reported to be evolutionarily closer tohuman cytomegalovirus than to Betaherpesviruses 6and 7 (Rawlinson et al. 1996; Vink et al. 2000). Themain conclusions of this study, therefore, do notchange significantly. For example, the number of func-tions shared within the Betaherpesvirus lineage is un-likely to be significantly different, as these are thegenes that the cytomegalovirus and the HHV-6/HHV-7branches share among each other. Another herpesviruscomplete genome that was not included is that of thechannel catfish herpesvirus, as this virus is a very dis-tant relative to the Alpha-, Beta-, and Gammaherpes-viruses (McGeoch and Davison 1999a).

During the preparation of this paper, a cross-genome comparison of gene content applied to a morerestricted subset of herpesvirus genomes (13) was pub-lished (Montague and Hutchison 2000). As in the pres-ent analysis, sequence similarity was initially detected

by BLASTP (Altschul et al. 1990), but families were con-structed by a different procedure and different strin-gency levels were tested. At the lowest stringency level,the authors detected 104 multiprotein families, a resultthat cannot be directly compared to our 243 familiesbecause our study includes more genomes (19). How-ever, the sensitivity of the two methods appears to bevery similar as the number of genes identified as con-served in all herpesvirus is essentially the same. Al-though the results appear consistent, the data pre-sented here provide a greater depth and insight intoherpesvirus phylogeny.

One of the objectives of this study was to establisha formal framework through the construction of ho-mologous families and phylogenetic profiles for thestudy of gene function in large families of viruses. Theproduction of a database of virus genomes and HPFs(VIDA, Virus Database) will greatly facilitate such fu-ture studies. This approach has proven useful in theinterpretation of herpesvirus homologous family con-tent and evolution and should also yield interestingresults when applied to other virus families. The futurecharacterization of new virus gene functions, togetherwith protein structure and gene expression data, willfurther strengthen the importance of genomewide in-tegrative approaches in the understanding of virusbiology.

METHODS

Identification of Homologous FamiliesA total of 19 complete genomes representative of viruses inthe Herpesviridae were retrieved from GenBank (see Table 1).Protein sequences from all identified ORFs were extracted andused to build up a protein-sequence dataset containing a totalof 1692 proteins. XDOM (Gouzy et al. 1997) was used to iden-tify homology between the proteins and to identify regions ofsequence similarity that were common to related proteins.XDOM is based on BLASTP (Altschul et al. 1990) and had pre-viously been used to identify regions of protein-sequencesimilarity in different complete genomes from bacteria, ar-chaea, and eukarya (Gouzy et al. 1999). Initially, we empiri-cally tested several parameters of the program so as to maxi-mize sensitivity without compromising accuracy. After theinitial observations, XDOM was used with the parametersSCORE = 75 and SCORE2 = 40 instead of the default values(90 and 50, respectively). We found that these parametersincreased sensitivity although they still prevented the appear-ance of spurious matches between functionally unrelated pro-teins. A C++ program, PSC BUILDER, was written to clusterprotein sequence domains together into HPFs. We clusteredall proteins that shared at least one sequence domain, so thatin each HPF there is at least one conserved region that ispresent in all proteins (Fig. 1). The method used identifies allproteins that share sequence similarity. Therefore, ortholo-gous and paralogous sequences, derived from recent gene du-plications, may be found in the same HPF. Proteins that didnot share sequence homology to any other protein weretreated as single-protein families. In these cases, the equiva-

Alba et al.


lent of the HPF-conserved sequence region will be the com-plete protein sequence.

Function IdentificationProtein function, if known, was extracted for each herpesvirusprotein from the original sequence-entry annotations. As nomajor disagreements were found in the annotated function ofdifferent proteins in the same homologous family, we consid-ered that a function could be used to define most herpesvirusHPFs. Functions were simple definitions such as DNA poly-merase or capsid protein. All protein functions were classifiedinto seven major pathways or functional classes: replication,nucleotide metabolism and DNA repair, transcription, struc-tural (including capsid, tegument, and virus assembly pro-teins), glycoproteins, others (including proteins involved inhost-virus interactions such as immune modulation proteins),and unknowns.

Phylogenetic Profiles of the Homologous FamiliesPhylogenetic profiles can be defined from the presence orabsence of a HPF in each virus genome (Pellegrini et al. 1999).A matrix was constructed, which for each homologous family,the presence of proteins from each given genome was ex-pressed as 1 (presence) or 0 (absence). The matrix consisted of439 columns for the total of homologous families, includingthose with only one protein, and 19 rows for the number ofherpesvirus genomes. The presence of more than one proteinfrom the same genome in the same homologous family (pre-sumably due to paralogous genes) was not taken into accountfor the purpose of matrix construction. For the separate analy-sis of functional class conservation, the complete matrix wassplit into class submatrices. The number of shared gene func-tions across all genomes was determined as a whole number,representing all homologous families in which both genomeswere present and also as a percentage of the number of sharedfunctions.

Phylogenetic Analysis of Herpesvirus Genomeson a Functional BasisThe phylogenetic profiles were used to conduct phylogeneticanalysis of the different viruses. The different protein familiescan be considered as molecular function characters for whichthe different viruses are positive (1) or negative (0). The datawas bootstrapped 100 times using our own scripts and maxi-mum parsimony, and distance methods (neighbor-joining)were applied.

For the distance methods, two distance measures wereused: (1) Fraction of nonshared functions dx,y = 1�[(positivein X and in Y)/(minimum between total positives in X andtotal positives in Y)] and (2) fraction of dissimilar functionsdx,y = [(positive in X but not in Y) + (positive in Y but not inX)]/total of homologous families.

In both cases, a positive refers to a 1 in the matrix (pres-ence of a gene from the homologous family in that genome).

The first measure was previously used to build trees fromgene content in unicellular organisms (Snel et al. 1999); thesecond was chosen because it may better satisfy the propertyof additivity of distance (Rzhetsky and Nei 1993). We used theprograms NEIGHBOR and DNAPARS from the PHYLI8P package(Felsenstein 1993) for neighbor-joining and maximum parsi-mony methods, respectively. Consensus trees were derivedusing CONSENSE from the same package. The final trees weredrawn with TREEVIEW (Page 1996).

Phylogenetic Analysis Based on ProteinSequence AlignmentsWe used the 26 ORFs identified as homologous in all Herpes-viridae to construct a phylogeny based on sequence similarity.Alignments from a total of 28 conserved domains from the 26ORFs and derived with MKDOM (Gouzy et al. 1997) were con-catenated to form a single alignment of 8900 amino acids,including gaps. The alignment was bootstrapped 100 timesand distances were computed with CLUSTALX default metricbased on the Gonnet matrices (Benner et al. 1994) and cor-rected for multiple substitutions. Neighbor-joining trees wereconstructed using CLUSTALX (Thompson et al. 1997); UPGMAand maximum parsimony trees were constructed usingNEIGHBOR and PROTPARS, respectively, from the PHYLIPpackage (Felsenstein 1993). Consensus trees were obtainedwith CONSENSE from PHYLIP and trees visualized with TREE-VIEW (Page 1996).

ACKNOWLEDGMENTSWe thank Robin A. Weiss and Sylvia Nagl for their advice onthis project. This work is funded by the Biotechnology andBiological Sciences Research Council (BBSRC; M.A.) and theMedical Research Council (MRC; C.O. and P.K.).

The publication costs of this article were defrayed in partby payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 USCsection 1734 solely to indicate this fact.

REFERENCESAltschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J.

1990. Basic local alignment search tool. J. Mol. Biol.215: 403–410.

Andrade, M.A., Ouzounis, C., Sander, C., Tamames, J., and Valencia,A. 1999. Functional classes in the three domains of life. J. Mol.Evol. 49: 551–557.

Benner, S.A., Cohen, M.A., and Gonnet, G.H. 1994. Amino acidsubstitution during functionally constrained divergent evolutionof protein sequences. Prot. Eng. 7: 1323–1332.

Benson, D.A., Boguski, M.S., Lipman, D.J., Ostell, J., Ouellette, B.F.,Rapp, B.A., and Wheeler, D.L. 1999. GenBank. Nucleic Acids Res.27: 12–17.

Cha, T.A., Tom, E., Kemble, G.W., Duke, G.M., Mocarski, E.S., andSpaete, R.R. 1996. Human cytomegalovirus clinical isolates carryat least 19 genes not found in laboratory strains. J. Virol.70: 78–83.

Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package), version3.5c. Distributed by the author. Department of Genetics,University of Washington, Seattle.

Fitz-Gibbon, S.T. and House, C.H. 1999. Whole genome-basedphylogenetic analysis of free-living microorganisms. Nucleic AcidsRes. 27: 4218–4222.

Gouzy, J., Eugene, P., Greene, E.A., Kahn, D., and Corpet, F. 1997.XDOM, a graphical tool to analyse domain arrangements in anyset of protein sequences. Comput. Appl. Biosci. 13: 601–608.

Gouzy, J., Corpet, F., and Kahn, D. 1999. Whole genome proteindomain analysis using a new method for domain clustering.Comput. Chem. 23: 333–340.

Hannenhalli, S., Chappey, C., Koonin, E.V., and Pevzner, P.A. 1995.Genome sequence comparison and scenarios for generearrangements: A test case. Genomics 30: 299–311.

Karlin, S., Mocarski, E.S., and Schachtel, G.A. 1994. Molecularevolution of herpesviruses: Genomic and protein sequencecomparison. J. Virol. 68: 1886–1902.

McGeoch, D.J. and Cook, S. 1994. Molecular phylogeny of theAlphaherpesvirinae subfamily and a proposed evolutionarytimescale. J. Mol. Biol. 238: 9–22.



McGeoch, D.J. and Davison, A.J. 1999a. The molecular evolutionaryhistory of herpesviuses. In Origin and Evolution of Viruses. LondonAcademic Press, UK.

———. 1999b. The descent of human herpesvirus 8. Seminars inCancer Biology 9: 201–209.

McGeoch, D.J., Cook, S., Dolan, A, Jamieson, F.E., and Telford,E.A.R. 1995. Molecular phylogeny and evolutionary timescale forthe family of mammalian herpesviruses. J. Mol. Biol.247: 443–458.

Montague, M.G. and Hutchison III, C.A. 2000. Gene contentphylogeny of herpesviruses. Proc. Natl. Acad. Sci. 97: 5334–5339.

Page, R.D.M. 1996 TREEVIEW: An application to displayphylogenetic trees on personal computers. Comput. Appl. Biosci.12: 357–358.

Pellegrini, M., Marcotte, E.M., Thompson, M.J., Eisenberg, D., andYeates, T.O. 1999. Assigning protein functions by comparativegenome analysis: Protein phylogenetic profiles. Proc. Natl. Acad.Sci. 96: 4285–4288.

Rawlinson, D.W., Farrell, H.E., and Barrell, B.G. 1996. Analysis of thecomplete DNA sequence of murine cytomegalovirus. J. Virol.70: 8833–8849.

Rzhetsky, A. and Nei, M. 1993. Theoretical foundation of theminimum-evolution method of phylogenetic inference. Mol. Biol.Evol. 10: 1073–1095.

Snel, B., Bork, P., and Huynen, M.A. 1999. Genome phylogeny basedon gene content. Nat. Gen. 21: 108–110.

Tatusov, R.L., Koonin, E.V., and Lipman, D.J. 1997. A genomicperspective on protein families. Science 278: 631–637.

Teichmann, S.A. and Mitchison, G. 1999. Making family trees fromgene families. Nat. Gen. 21: 66–67.

Tekaia, F., Lazcano, A., and Dujon, B. 1999. The genomic tree asrevealed from whole proteome comparisons. Genome Res.9: 550–557.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., andHiggins, D.G. 1997. The ClustalX windows interface: Flexiblestrategies for multiple sequence alignment aided by qualityanalysis tools. Nucleic Acids Res. 24: 4876–4882.

Vink, C., Beuken, E., and Bruggeman, A. 2000. Complete DNAsequence of the rat cytomegalovirus genome. J. Virol.74: 7656–7665.

Received May 31, 2000; accepted in revised form October 26, 2000.

Alba et al.


genomewide function conservation and phylogeny in the

Documents