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JOURNAL OF VIROLOGY,0022-538X/00/$04.000

Aug. 2000, p. 70797084 Vol. 74, No. 15

Copyright 2000, American Society for Microbiology. All Rights Reserved.

A Hypothesis for DNA Viruses as the Origin of EukaryoticReplication Proteins

LUIS P. VILLARREAL1* AND VICTOR R. DEFILIPPIS2

Departments of Molecular Biology and Biochemistry1 and Ecology and Evolutionary Biology,2

University of California, Irvine, California 92697

Received 16 December 1999/Accepted 1 May 2000

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic andbacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virusreplication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase fromphycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitiveeukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes andcertain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presenceof highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetictree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryoticDNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We

consider arguments for the polarity of this relationship and present the hypothesis that the replication genesof DNA viruses gave rise to those of eukaryotes and not the reverse direction.

Divergence of the bacterial and eukaryotic lineages appearsto represent the deepest split in the tree of life (22). Becausethe DNA replication proteins of these groups are of funda-mental importance and interact through complex mechanisms,it seems likely that the genome replication system, like thetranslational system, would contain the most conserved co-evolved genes among all related lineages.

Obvious functional homologues of replication genes arefound in bacteria, eukaryotes, and archaea, including proteinsinvolved in origin recognition, helicases, DNA-binding pro-teins, DNA synthesis, sliding clamp processivity factors

(PCNA), ligation, and primer removal (see reference 7 andreferences therein). However, there are clear differences insequence similarity that separate the replication proteins ofbacteria from those of the archea and eukaryotes (7). Thebacterial replication genes thus appear evolutionarily unre-lated to those of eukaryotes and archaea. For example, thereplicative DNA polymerase (Pol) III of Escherichia coli be-longs to the family C DNA Pol group and does not havesimilarity to either of the two mammalian replicative DNAfamily B DNA Pols (alpha priming and delta extending; seereference 30). As such, phylogenetic analysis of these replica-tive DNA Pols results in polyphyletic groupings that are con-trary to accepted species trees (6). Such wide existence offunctionally identical yet nonorthologous genes presents a di-

lemma when they are being used for connecting the universaltree of life, and this has led some to propose that the cenan-cestor of bacteria, archea, and eukaryotes had an RNA ge-nome (7, 17). However, it is now clear that between bacteriaand eukaryotes, perhaps several hundred functional genes arehomologous (e.g., DNA synthesis genes). This suggests thatthe putative prokaryotic-eukaryotic ancestor possessed manygenes inherited by both lineages (for references, see references14 and 8). Proper replicative transmission of such a largenumber of essential genes seems unlikely given the small size

of RNA genomes and the error-prone nature of their replica-tion (14). It therefore appears more likely that the commonancestor had a DNA genome, which leaves unexplained howthe replication systems underwent the transition during thedivergence of bacteria from archaea and eukaryotes.

DNA viruses, however, also possess a full set of independentDNA replication and repair proteins that include members offamily A and B DNA Pols (12). When first sequenced, it wasnoteworthy how similar phage T4 DNA Pol was to DNA Polsalpha and delta of eukaryotes, Epstein-Barr virus, human cy-tomegalovirus, and other DNA viruses of eukaryotes, but notadenoviruses or E. coli Pol I or III (23). This similarity includesthe conservation of five of six sequential domains (31), as wellas resistance to various family B-specific inhibitors (3). Otherphage DNA Pols, however, such as T7, show similarity tobacterial DNA Pol I but not to Pols of eukaryotes. With thesequencing of the entire T4 genome, it was additionally sur-prising to see that this strictly lytic bacteriophage had moregenes similar to those of eukaryotes (including genes for self-splicing RNA [13]) than to bacterial genes (4). Viruses areusually thought to impose negative selection on their hosts. Inaddition, recombination between host and viral genomes is acommonly observed phenomenon, such as with retrovirusesacquiring cellular protooncogenes (5, 28). Yet viruses arerarely considered a source of host genes, and hence viral se-

quences are not taken into account when reconstructing thetree of life. However, a viral genome can evolve up to a milliontime faster than that of its host. If a DNA virus could imposea stable persistent (or genomic) infection on its host, it mightthen also provide genes altering host evolution, as we havepreviously reasoned (29). This raises the question: Could aDNA virus have been the origin of replicative eukaryotic DNAPols?

In this report, we consider the hypothesis for the viral originof eukaryotic replication proteins in the context of DNA vi-ruses that infect host species which are likely representative ofthe earliest eukaryotes. We examine DNA Pols from two fam-ilies of DNA viruses prevalent as acute infections of parasiticmicroalgae (Chlorella-like viruses) (27) and persistent infec-tions of filamentous brown algae (Feldmania species virus) (9,

* Corresponding author. Mailing address: Department of MolecularBiology and Biochemistry, 3205 Bio Sci II, University of California,Irvine, Irvine, CA 92697. Phone: (949) 824-6074. Fax: (949) 824-8551.E-mail: [email protected].

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15, 16, 21, 27). These algal species represent some of theearliest eukaryotes for which clear archaeological data exist(11). We perform sequence similarity and phylogenetic analy-ses which indicate that these viral proteins appear related tothe progenitor of all eukaryotic Pol delta sequences and con-sider arguments that a DNA virus may have been the origin ofthe eukaryotic DNA replication system.

MATERIALS AND METHODS

The open reading frame that codes for the DNA Pol or Pol-like gene fromChlorella virus (NT2A; GenBank M86836; 913 amino acids [a.a.]) and Feldmaniaspecies virus (GenBank AF013260; 996 a.a.) were retrieved from GenBank.Using these sequences, a gapped Tblastn (version 2.0.4) analysis against thetranslated nonredundant database was performed. It was observed that essen-tially all of the replicative DNA family B Pols from eukaryotes showed similarityto both sequence probes. In addition, the DNA Pol sequences from most largeDNA viruses of animals were also identified. Although the analysis suggests thatall eukaryotic replicative DNA Pols (alpha and delta) are similar, the DNA Poldelta genes were most similar to these phycodnavirus-like genes. Interestingly,although Feldmania virus and Chlorella virus are both DNA viruses of algae, eachof these DNA Pol sequences was more similar to a lower eukaryotic host DNAPol gene (Schizosaccharomyces pombe, Candida albicans, Glycine max, or Sac-charomyces cerevisi ae) than to each other. In addition, the DNA Pols of severallytic phages (T4 and RB69) were identified. Also present were the DNA Pol II

genes from various archaebacterial and bacterial (i.e., nonreplicative E. coli)species. Absent were the replicative DNA polymerases (Pol III) and Pol I frombacteria as well as the DNA Pols of other lytic phages (T7), adenoviruses, andrelated linear plasmids of fungi.

Following the elimination of redundant and incomplete proteins, the remain-ing sequences were aligned using ClustalW to aid in identification of homologousregions. After this alignment, four regions (labeled I, II, III, and IV) of highconservation were easily identifiable between most of the taxa and are shownlisted in color patterns corresponding to similar amino acids and in biologicallyrelated groups (Fig. 1). As had previously been established, the family B poly-merase sequences contain up to six specific domains (23, 31). We compared ourconserved domains to those previously identified and determined that our re-gions II, III, and IV corresponded roughly to the respective regions II, III, andIV which were identified in DNA Pol alpha by Wang et al. and that our regionI had been previously identified as the phosphonoacetic acid-resistant domain ofherpes simplex virus type 1 DNA Pol in the study to T4 DNA Pol by Spicer et al.(23). Because there is large variation in length among these DNA Pol genes, thesequences are shown as a roughly proportional line drawing in which the loca-tions of the four highly conserved domains are indicated, and the sequences were

centered to the most highly conserved region II domain (Fig. 2). The twosmallest sequences correspond to fragments of Micromonas pusilla virus andChrysochromulina species virus (phycodnavirus). The next largest was the fullgene (313 a.a.) for the Pol alpha of Endotrypanum (Leishmania) monterogeni,then the Helicoverpa armigera nuclear polyhedrosis virus DNA Pol (623 a.a.), andall other genes were complete sequences. The largest gene (encoding 1,855 a.a.)was the DNA Pol alpha ofPlasmodium falciparum. In general, domains I and IIare adjacent to each other and occur at variable positions from the aminoterminus, although some Archaea species Pol II genes have a region I domainwell displaced toward the amino terminus. With the exception ofHalteria speciesDNA Pol alpha (ciliated hypotrichous), the order of the domains was conserved,although DNA Pol alpha genes of hyptrochous species were often lacking do-mains II and IV. In addition, the DNA Pol II of several archaea (lineage A) haddomains III and IV displaced well towards the carboxy terminus.

These highly conserved regions were then used to aid in the alignment of theremaining regions as follows. First, using the sequence editor GeneDoc version2.5 (18), each taxon was examined to determine which if any of the four domainswere present in the protein sequence. Next, these regions were used as anchors

from which to optimize the alignment of amino acids in the intervening sections.These interregion sequences were extracted and aligned using ClustalW. Fol-lowing this procedure, the alignments were again optimized by eye, focusingmostly on the similarity within each of the major clades. Once an overall align-ment was obtained, a phylogenetic tree was constructed using the more con-served amino terminus of the protein sequence that included region I and aminoacids thereafter. Phylogenetic analysis was performed using the neighbor-joiningalgorithm with 500 bootstrap replications (20) as implemented by PAUP version4.0b2 (25). Pairwise distances were calculated as mean observed substitutions persite. The unrooted tree is shown in Fig. 3 and is color coded to mark clear clades.

RESULTS

The results suggest that the relationships are robust: 68% ofthe nodes had 90% bootstrap frequency support, and allnodes were 50%. The unrooted tree shows DNA Pol se-quences falling into seven clades that correspond to biologi-cally coherent gene sets. The two largest clades correspond to

variants of DNA Pol alpha (pink) and DNA Pol delta, respec-tively. In the DNA Pol delta clade (black), the Feldmaniaspecies virus (which causes a prevalent persistent infection offilamentous brown algae) DNA Pol is near the base (labeledpol delta) and the Chlorella-like viral Pol genes are slightlymore derived. Other Pol delta proteins appear to correspondroughly with accepted evolutionary relationships. The topologyof the DNA Pol alpha group is more complex. Near its root,the trypanosomes and Leishmania species branch first, fol-lowed by insects and mammals, which, interestingly, aregrouped separately from Saccharomyces and Schizosaccharo-myces pombe. Also branching near the base of this clade arethe macronuclear genes of various binucleated hypotrich spe-cies.

There are three distinct clades of viral DNA Pols. Two ofthese correspond to the poxvirus family (light gray) and thebaculoviruses of insects that includes the nucleopolyhedrosisvirus family (green). Both of these groups branch from themost unresolved region at the center of the tree. The thirdclade corresponds to the animal herpesviruses (red). It is in-teresting that the herpesviruses appear to share an ancestorwith the Feldmania DNA Pol, which corresponds to the base ofthe cellular DNA Pol delta clade. The herpesviruses are fur-ther branched into three monophyletic subgroups correspond-ing to the alphaherpes-, gammaherpes-, and cytomegalovi-ruses. The placement of the herpesvirus ancestor near theunresolved center of the tree suggests a very old origin of thesegenes.

The remaining two groups include the replicative DNA Pol

II genes from various archaea (methanogens and Thermococ-cus, Pyrococcus, and Sulfolobus species), which were known tobe similar to family B DNA Pols (19). DNA Pol II of archaeaspecies appears to exist as two distinct lineages, both of whichare thought to be involved in genome replication (7, 26). Thelarger of these groups appear to share an ancestor with theDNA Pol alpha genes (blue). The smaller clade (gold) corre-sponds to DNA Pols found in Solfolobus and pyrodiococciarchaea species. The archaeal DNA Pols on this smallerbranch are closer but not directly connected to the Pol deltagroup. This cluster is rooted near the unresolved center of thetree. Also originating near the unresolved center are the Polsfrom lytic phages T4 and RB69 and from E. coli DNA Pol II(nonessential Pol).

DISCUSSION

With sequences obtained from a similarity search usingDNA Pols from DNA viruses that infect microalgae and fila-mentous brown algae as a probe, we generated a phylogeny inwhich the base of the monophyletic group containing the rep-licative DNA Pol delta of eukaryotes resembles viral se-quences. Although an earlier analysis of DNA Pol genes gave

FIG. 1. Amino acid alignment of four highly conserved DNA Pol protein regions. Taxon names are color coded according to clade as in Fig. 3 and are labeled A0to L5 according to the branch tips therein. Gaps inserted to improve the alignment are indicated by a dash (). Amino acids are color coded according to side groupproperties using the following scheme: red, negatively charged (D or E); orange, positively charged (H, K, or R); light green, amide (N or Q); blue, alcohol (S or T);purple, aliphatic (L, I, or V); gray, aromatic (F, Y, or W); brown, small (A or G); dark green, sulfur-containing (M or C); white, proline (P). Abbreviations: Hu, human;VZV, varicella-zoster virus; HSV, herpes simplex virus; cytomeg., cytomegalovirus; HHV, human herpesvirus.

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rise to similar patterns, the authors did not attempt to explainthis result (6). Since it is unrooted, the phylogeny does notdirectly establish the polarity or direction of evolutionarychange. It therefore remains formally possible that the phy-codnaviruses acquired DNA Pol genes from their algal hostsand maintained similarity to them for unknown reasons. As the

algal host DNA Pol genes have not been sequenced, we cannotplace them on this tree. Even if they were subsequently to beplaced phylogenetically near the phycodnavirus genes, thiswould still be unlikely to resolve the issue of evolutionarydirection. However, we believe several considerations arguethat the direction of transmission was from virus to host. First,only under this circumstance could the dilemma of dissimilarreplication genes now present in bacteria and eukaryotes beresolved. In addition, all the other viral DNA Pols examinedform distinct monophyletic groups (i.e., herpesviruses, poxvi-ruses, and baculoviruses) that do not include host Pols. There-fore, these other viruses did not appear to acquire their Polgenes from a host species. The DNA delta clade is clearlymonophyletic yet includes all the diverse phycodnavirus Pols ofboth microalgal and filamentous algal hosts. Thus, the phycod-

naviruses are clearly evolutionarily exceptional DNA viruses.The simplest way to account for these observations is to pro-pose that host Pol delta genes are derived from an early DNAviral gene that resembles that present in Feldmania virus.

Trees of life have been generated using different genes,yielding multiple evolutionary histories (8). Phylogenetic anal-

ysis of DNA Pol sequences presents patterns inconsistent withaccepted organismal phylogenies. These phylogenetic dispari-ties are difficult to explain if most genetic variation duringevolution of species occurs by random genetic change andvertical gene transmission. Genomic analysis has suggestedthat horizontal transfer of gene sets may have been moreprevalent then previously believed, especially in bacterial spe-cies. Horizontal transmission of DNA replication genes, how-ever, would suggest the transfer of fundamental, complex, cel-lular components and the involvement of a DNA virus. Wehave argued that the persistence of a genetic parasite (a virusor its defective derivatives) is a life strategy that can allow thesuperimposition of complex molecular genetic control systemsonto its host (29). As such, a persistent agent (like Feldmaniavirus) can potentially provide new systems of genetic control,

FIG. 2. Protein map indicating proportional lengths of DNA Pol (black lines) and relative locations of the four conserved Pol protein domains (labeled I to IV).Proteins are mostly centered so that region II is aligned.

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including genome replication, to its host, particularly if it isintegrated into the genome. We suggest at least in the case ofDNA Pol delta an evolutionary link of the bacteria and eu-karyota (and archaea) via the DNA Pol of an ancient DNAvirus, not the replicative host genes. Our analysis also suggeststhat DNA Pol alpha may share an ancestor with DNA Pol II ofarchaea that diverged after the initial divergence of bacteriafrom eukaryotes and archaea. Two other DNA Pols resemblethe family B replicative Pols of eukaryotes and archaea. One is

the nonessential Pol II of E. coli, and the other is the Pol fromlytic phages T4 and RB69. Both branch from the largely un-resolved center of the tree. As the phages represent a muchmore transmissible system than E. coli Pol II, and as T-likephages infect both bacteria and archaea (Euryachaeota king-dom [32]), it is easier to envision substitution of functionalhomologues for DNA replication genes if such a virus wasinvolved. Other DNA replication genes may also fit this pat-tern, since it is known that DNA viruses also code for variousligases, helicases, and PCNA-like genes as well as repair-likeDNA Pols, such as DNA Pol beta, found in entomopoxvirus(1).

Many of the crucial regulatory genes of DNA viruses, suchas the T antigens of polyomaviruses, have no known host an-alogues, even though these viruses are phylogenetically con-

gruent with their host species over long periods of time (29).Thus, at least for these regulatory genes, they are viral, nothost, creations. Viral genomes can evolve much faster thanhost genomes, and populations are known to exhibit muchgreater genetic variability, as demonstrated by the frequentoccurrence of mutants and defectives. Thus, viral systems havean enhanced capacity to produce genetic novelty. Althoughsome examples of virus-mediated horizontal gene transferhave recently been proposed (2), in most of these proposals it

is suggested that the host, not the virus, is the original sourceof the transferred gene. We now suggest that such infectiousand/or persisting agents may be a general source for acquisi-tion of complex molecular systems and phenotypes.

ACKNOWLEDGMENT

This research was supported by the Irvine Research Unit in AnimalVirology.

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FIG. 3. Unrooted neighbor-joining phylogeny based on amino-terminal portion of DNA Pol protein sequences as discussed in the text. Labels at branch tipsrepresent taxa as presented in Fig. 1. Numbers at branch nodes indicate percent bootstrap support for that node based on 500 replications.

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