Luis P. Villarreal is Professor of Molecular Biology and Biochemistry at the University of California Irvine. This article is based on a presentation at the annual meeting of the American Society for the Advancement of Science in January 2001.
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Persisting Viruses Could Play Role in Driving Host Evolution
Genetic parasites may be the legacy of punctuated events during evolution, including eukaryotic replication and immune tolerance
Luis P. Villarreal
When a persistent virus or its defective counterpart colonizes a host, it can have major consequences, perhaps enabling the host to rapidly acquire a complex phenotype. This issue of complex phenotype acquisition, which requires coordinated expression of several complementing or interacting genes, raises challenging questions for evolutionary biologists to address. Such events seem to mark major breaks and new orders, such as development of the eukaryotic nucleus or of the adaptive immune system. Yet, these punctuated acquisitions would not appear likely or even feasible based on conventional neo-Darwinian models for evolution that emphasize point mutations and sexual recombination of host genes to create novel phenotypes.
However, these models ignore well-established microbial phenomena. For instance, in a single step, a bacterium can acquire a full set of genes conferring virulence or multiple drug resistance. Although such events may involve horizontal transfers of genes by transposons, they also may be mediated by phage following lysogenic infections—for example, prophage V cholera, which actually involves two distinct phages. Moreover, some apparent transpositions closely resemble transfers mediated by defective viruses and could well derive from phage-mediated transfers, according to Allan M. Campbell, Stanford University, Calif. Indeed, by comparing sequence data for the Escherichia coli and Bacillus subtilis genomes, we find that they differ in about 230 regions and that these differences are mostly adjacent to tRNA sites, suggesting sites of prophage integration instead of transposon-mediated transduction.
Phage Appear To Mediate Bacterial Evolution
Thus bacteria, the most adaptable of organisms, evolve in part by infectious, phage-mediated processes that involve persisting parasitic genomes occupying host enomes and introducing novel phenotypes. Although many microbiologists continue to call this "horizontal transmission," implying that viruses serve merely as vehicles for moving genes between two cellular hosts, I argue that bacteria derive many, if not most, of their novel gene sets from viral, not other, host sources. Given the higher relative rates of viral variation, recombination, and adaptation, viruses rather than bacteria can massively explore sequence space.
For example, T4 phage has 225 genes, only 69 of which are essential for growth in E. coli and only 42 of which are similar to genes in GenBank. The majority of these GenBank-related genes are curiously more related to eukaryotic genes, including the self-splicing group I introns (discovered in the T4 TK gene). Other viruses are similar in their degree of novelty while lacking substantial similarity to host genes, including most eukaryotic DNA viruses. Thus, the large majority of phage and viral genes are unique to various families of virus, not hosts, although some striking similarities to eukaryotic genes exist.
To persistently occupy its host, a virus needs to be very competitive, and this often entails very specific components. For example, the RIIA gene of T4, used to elucidate the most basic molecular aspects of genes, has no required function within its host, E. coli. Instead, this viral gene is needed solely to enable T4 to infect an E. coli cell that already is occupied by a lysogen. In fact, other T-even phages carry highly conserved, early genes of unknown function that apparently are not necessary for viral replication. Furthermore, these genes generally have no host analogs, suggesting that they were not "stolen" from host genomes as some phage experts assert.
The Origin of Eukaryotes
If bacteria evolved by means of infectious mechanisms, were similar mechanisms at work for eukaryotes? Are we overlooking examples of infection- or transduction-mediated genetic adaptation? Could persisting parasitic genetic agents somehow be involved in eukaryotic evolution? Or do complex structures and physiologic processes arise in eukaryotes solely by means of point mutations, sexual exchanges, and recombination?
Many metazoan organisms carry a load of parasitic genetic elements—often called junk DNA—that presumably accumulated steadily during evolution. Comparing E. coli with humans, we see only a surprisingly modest increase in gene number from 2,350 to about 40,000. We also see gene density drop from 90% coding sequence within the genome of E. coli to less then 2% within the human genome, which is replete with noncoding, parasitic elements, including type I and II transposons, and other distinctive parasitic elements, such as long and short interspersed elements (LINEs and SINEs). The genomes of humans and other vertebrates also contain several apparently intact endogenous retroviruses.
For example, human chromosome 21 carries 225 protein-encoding genes, but also carries 2,000 endogenous retroviral elements. In addition, investigators participating in the human genome project identified 113 examples of human genes that are not found in several other simpler eukaryotes, but can be found in bacterial species—which is consistent with direct horizontal transfers of genes between bacteria and vertebrates.
The biggest discontinuity in evolution—between bacteria and eukaryotes—is reflected in differences between their respective replication proteins. Both bacteria and eukaryotes use specialized sets of proteins to replicate their DNA. Although these complex, highly interacting sets of proteins perform very similar functions, they have few or no sequences in common and show no evidence of deriving from a common ancestor. Moreover, although replication proteins from eukaryotes share some features with their counterparts among archebacterial replication proteins, the two sets also differ markedly, including in terms of DNA-origin recognition complexes and single-stranded DNA-binding proteins.
Striking Similarities between Certain Phage and Eukaryotic Genes
However, there are some striking similarities between certain phage and eukaryotic genes. For example, the DNA polymerase gene of T4 phage is very similar to that of eukaryotes. The T4 DNA polymerase is a member of the Pol Beta family, sensitive to the same inhibitors as eukaryotic DNA Pol delta (extension) and alpha (primase) polymerase, leading Margarita Salsa and Luis Blanco of the Universidad Autonoma, Madrid, Spain, to suggest a common origin for them. T4 is a tailed phage, much like those also found in archaebacteria (e.g., the H phage) and cyanobacteria.
Although T4 is strictly lytic in E. coli, distantly related viruses can infect some algae species. Some of these viruses, such as the Feldmania species virus, are persistent and inapparent, whereas others, such as Chlorella species virus, CSV-1, of microalgae, are lytic. Chlorella species are unicellular, parasitic, haploid, and asexual. CSV-1 is a double-stranded DNA virus (380 kbp), whose genes encode a DNA delta-like polymerase, two proliferating cell nuclear antigen (PCNA)-like genes, thymidine kinase, 10 tRNA molecules, tRNA synthase, superoxide dismutase, several versions of ribonucleotide reductase, 12 restriction and modification enzymes (rare in eukaryotes), hyaluronic acid synthase, cellulose synthase, and a bacterium-like transposase; the genome also contains about 80 introns. The evolutionary junction between microalgae and the filamentous brown algae also coincides with distinctions that developed after the Precambrian radiation, when multicellular metazoans and sexual reproduction emerged.
Could a persisting DNA virus of algae have been the origin of the eukaryotic replication proteins and thus connect the universal tree of life via a viral linkage? To examine this possibility, we used the TBLASTN program to compare the DNA polymerase (Pol) sequence of Feldmania virus, which is specific to Feldmania species of filamentous brown algae, with a series of other Pols. Included were the DNA Pol genes from four families of DNA viruses (phycodnaviruses, herpesviruses [with alpha, beta, and gamma subclades], the poxviruses, and the baculoviruses), several bacterial phage, and two distinct sets of archebacterial DNA Pol IIs.
According to this analysis, essentially every member of the DNA polymerase B family is similar, with conserved functional domains among all these proteins. Furthermore, when analyzed by neighbor-joining methods with strong bootstrap statistical support, the unrooted dendogram identifies sets, or clades, of related polymerases. The clades correspond to coherent biological sets and include the delta extension Pol and the primase interacting Pol alpha of host cells.
Most of these clades are distinct from one another, and many of them link to the unresolved center of the tree—with one notable exception. The DNA Pol from Feldmania virus is located at the base of the clade that corresponds to that of the host cellular DNA Pol delta. Since such trees do not establish polarity, this result suggests two very different possibilities: either (1) the DNA Pol of Feldmania virus represents the progenitor of all cellular DNA Pol deltas, or (2) the Feldmania virus acquired its version of DNA Pol from a host that resembles that progenitor.
A viral progenitor seems more likely. All other viral DNA Pols within this analysis are not part of their corresponding host clades even though these viruses appear as old on the dendogram as Feldmania and are also related to phage. Also, if Feldmania virus obtained its Pol from its host, it would be unusual compared to other DNA viruses. Since viruses are transmissible, it seems simpler for the transfer having been from virus to host. Moreover, proposing that a virus was the original source of eukaryotic replication proteins could help to explain the discontinuity between bacterial and eukaryotic replication proteins, while it would also explain how eukaryotes are linked to the bacterial world. Patric Forterre of the Universite de Paris-Sud in Orsay, France, has made similar proposals.
We also analyzed other phycoadnavirus genes, specifically superoxide dismutase (SOD) and PCNA. Although the results are less compelling because the data sets are smaller and there is less robust bootstrap support, they are consistent with a viral origin for these genes.
Mammals May Use Endogenous Retroviruses To Suppress In Utero Immunity
Biologists face a major challenge in explaining how mammals with adaptive immune systems tolerate carrying a sexually produced allogeneic embryo and endure other complicated processes that are part of live births. This complex phenomenon is the defining phenotype for mammalian organisms, one that presumably arose abruptly.
All mammalian genomes have specific and distinct sets of endogenous retroviruses (ERV) and much greater numbers of defective retroviral derivatives, suggesting that mammalian genomes were colonized by specific lineages of ERV soon after placental species radiated from one another. Examples include the human LINE-1, SINE R, and more distant and numerous Alu, related to HERV-K, and the mouse-IAP, hamster-IAP (distinct from mouse), feline-Rd114, and rhesus-Mason-Pfizer virus. Retroposon and ERV nomenclature is confusing. The human genome project indicates that there are several thousand human ERVs, and they appear to comprise 24 families. Humans have both ancient, such as ERV-L, and newly acquired versions of ERVs, such as eight ERV K members, which distinguish humans from close primate relatives.
Meanwhile, avian species lack such an intense level of retroposon colonization, according to David Mindell of the University of Michigan, Ann Arbor. Furthermore, mammals are phylogenetically congruent with their ERVs, whereas birds are not. Mammals and birds also differ in that although their early embryos are both susceptible to genomic infection with retroviruses, mammals repress these viruses (via global DNA methylation) in embryonic tissue. Although the more defective derivatives of these ERVs (especially whose env gene is deleted) do not usually encode for products, synonymous codon analysis indicates that the much smaller number of intact ERVs are maintained along with coding potential.
Mammalian species develop a trophectoderm-derived placenta, which enables the fertilized egg to invade and implant the uterine wall, establishes blood contact across the uterine wall, controls hormonal levels during gestation, and also protects the embryo from the mother's innate and adaptive immune response. The trophectoderm is the first cell to differentiate from the fertilized egg at 3.5 days when the totipotent inner cell mass (embryonic stem cells) is established.
In the 1970s, researchers observed that normal human and other mammalian trophectoderm or placental cells produce endogenous retroviral particles in large numbers. Later, others discovered that most mammals express their corresponding ERVs in placental and embryonic tissues. Furthermore, the human ERV sequences being expressed or transcribed in the early embryo and placenta are diverse, with some of them intact and others defective. Several ERV protein products are produced, including various ones containing env sequences. Placental and early embryo tissues are by far the most common and abundant sites of ERV expression, followed by lymphatic and malignant tissues.
Erik Larsson and colleagues at Uppsala University in Sweden observed ERV-3 env expression in normal human placental cells and suggest that intact ERV-3 is needed, possibly for immune suppression. I subsequently generalized this concept, taking into account the widespread colonization of placental species by intact ERVs and the general immunosuppressive nature of the ERV env membrane-spanning region. Although ERV-3 env is mutated in 1% of Caucasians, the complexity and number of other human ERV family members suggest that this and other ERV env proteins are being expressed and could be involved in immune suppression and other vital developmental processes.
Testing the Idea that ERVs Protect Mammalian Embryos
The idea that ERVs are somehow involved in protecting mammalian embryos is plausible and attractive. But testing this idea by suppressing all ERV families in early embryos is daunting, particularly because there are thousands of ERV loci.
However, findings from polyomavirus studies during the late 1970s provided us a means for addressing these questions. Those studies indicate that when embryonic carcinoma (EC) cells express the large T antigen (T-Ag) of the SV40 polyomavirus, it blocks expression of endogenous retroviruses without affecting EC cell differentiation. Moreover, T-Ag is expressed in embryonic stem (ES) cells without affecting their differentiation. However, transgenic lineages fail to establish, suggesting to us that T-Ag expression disrupts ERV expression without affecting cellular differentiation.
To test this idea, Alex Espinosa and I choose to selectively alter gene expression in mouse embryos by looking at EC cells because ES cells, although preferable in some respects, are no longer capable of differentiating into trophectoderm. Thus, we used F9 EC cells to evaluate the affect of T-Ag on expression of IAPE-A, a mouse ERV that expresses an env protein. Indeed, this env sequence is highly expressed in a normal mouse blastocyst. However, in EC cells, T-Ag prevents expression of IAPE-A without affecting EC differentiation into embryonic bodies, which closely resemble 3.5-day blastocysts. Although EC-derived embryonic bodies are not fully functional and cannot produce viable offspring (due to lost totipotency), they undergo implantation. However, the T-Ag-expressing embryonic bodies fail to implant.
These results are consistent with ERVs playing a role during embryo implantation, perhaps enabling embryos to avoid recognition by the mother's immune system. Although these results are not definitive, the implication is that placental mammals may have indeed evolved via the colonization by endogenous retroviruses that bestow complex phenotypes onto the embryos, especially via their placentas.
Thus, placental embryos apparently behave like parasites that invade and infect their hosts—namely, their mothers—drawing sustenance and producing local viruses to suppress host immune responses. This relationship resembles a phenomenon observed among some wasp species that implant their eggs into a larval caterpillar host. The host's innate defenses are neutralized by endogenous genomic viruses (polyadnaviruses) made within wasp nurse cells that surround the egg and block the caterpillar host's antiparasite defense responses. From these infected host larvae hatch flying wasps, a very distinct morphological life form. Thus, it is not so farfetched to think that such parasitic mechanisms might be the very basis for morphogenesis, so common to many flying insects.
As we consider other events during evolution in which organisms have acquired complex and highly adaptive phenotypes, we should look for the footprints of persistent genetic parasites. After all, the RAG1 and RAG2 recombinases, which are essential to the origin and function of the adaptive immune vertebrate system, closely resemble a retroviral integrase. The major histocompatibility complex locus itself is suspiciously colonized with 10 times the usual genomic frequency of ERVs. These and other footprints may well be the legacy of viruses that helped to make us.
SUGGESTED READING
DeFilippis, V. R., and L. P. Villarreal. 2000. A hypothesis for DNA viruses as the origin of eukaryotic replication proteins. J. Virol. 74:7079-7084.
Espinosa, A., and L. P. Villarreal. 2000. T-Ag inhibits implantation of EC derived embryoid bodies. Virus Genes 20:195-200.
Forterre, P. 1999. Displacement of cellular proteins by functional analogues from plasmids and viruses could explain puzzling phylogenies of many DNA informational proteins. Mol. Microbiol. 33:457-465.
Villarreal, L. P. 1999. DNA viruses contribution to host evolution, p. 391-419. In E. Domingo, R. Webster, J. Holland, and T. Picknett (ed.), Origin and evolution of viruses. Academic Press, London.
Villarreal, L. P. 1997. On viruses, sex and motherhood. J. Virol. 71:859-865.
Villarreal, L. P., V. R. Defilippis, and K. A. Gottlieb. 2000. Acute and persistent viral life strategies and their relationship to emerging disease. Virology 272:1-6.
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