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1.  The Extraordinary Evolutionary History of the Reticuloendotheliosis Viruses 
PLoS Biology  2013;11(8):e1001642.
Reticuloendotheliosis viruses are mammalian retroviruses that were transmitted to avian hosts through inadvertent human intervention, and subsequently integrated their genetic material into the genomes of large DNA viruses, generating novel recombinant pathogens that now circulate naturally in poultry and wild birds.
The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs—unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events.
Author Summary
Retroviruses are characterized by their ability to insert a DNA copy of their genome into the chromosomes of infected cells. Occasionally, retroviruses insert into “germline” cells and are subsequently inherited as host alleles called endogenous retroviruses (ERVs). Vertebrate genomes contain thousands of ERV sequences derived from ancient retroviruses, and these viral sequences serve as molecular “fossils” that can be used to explore how retroviruses have evolved over millions of years. Here we combine an analysis of the retroviral “fossil record” with a phylogenetic and historical investigation to determine the origin of a group of avian retroviruses called reticuloendotheliosis viruses (REVs). We present evidence to demonstrate that rather than arising from natural infections of birds, REVs are in fact derived from mammalian retroviruses that were accidentally introduced into avian hosts during experimental studies of a malaria parasite in the late 1930s. Remarkably, REVs have subsequently inserted into the genomes of two large DNA viruses that infect birds, generating chimeric viruses that now circulate naturally in poultry and wild birds.
PMCID: PMC3754887  PMID: 24013706
2.  A Paradigm for Virus–Host Coevolution: Sequential Counter-Adaptations between Endogenous and Exogenous Retroviruses 
PLoS Pathogens  2007;3(11):e170.
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5–7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (∼ 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.
Author Summary
The genome of all vertebrates is heavily colonized by “endogenous” retroviruses (ERVs). ERVs derive from retrovirus infections of the germ cells of the host during evolution, leading to permanent integration of the viral genome into the host DNA. Because ERVs are integrated in the host genome, they are transmitted to subsequent generations like any other host gene. The function of endogenous retroviruses is not completely clear, but some ERVs can block the replication cycle of horizontally transmitted “exogenous” pathogenic retroviruses. These observations lead to the hypothesis that ERVs have protected the host during evolution against incoming pathogenic retroviruses. Here, by characterizing the evolutionary history and molecular virology of a particular group of endogenous betaretroviruses of sheep (enJSRVs) we show a fascinating series of events unveiling the endless struggle between host and retroviruses. In particular, we discovered that: (i) two enJSRV loci that entered the host genome before speciation within the genus Ovis (∼ 3 million y ago) acquired, after their integration, a mutated defective viral protein capable of blocking exogenous related retroviruses; (ii) both these transdominant enJSRV loci became fixed in the host genome before or around sheep domestication (∼ 10,000 y ago); (iii) the invasion of the sheep genome by ERVs of the JSRV/enJSRVs group is still in progress; and (iv) new viruses have recently emerged (less than 200 y ago) that can escape the transdominant enJSRV loci. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.
PMCID: PMC2065879  PMID: 17997604
3.  Evidence for Restriction of Ancient Primate Gammaretroviruses by APOBEC3 but Not TRIM5α Proteins 
PLoS Pathogens  2008;4(10):e1000181.
Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5α and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a ‘fossil record’ of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5α proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species–dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.
Author Summary
Retroviruses integrate their genomes into host-cell DNA as an essential part of their replication cycle. If a retrovirus is integrated into a cell that becomes a germ line cell such as a sperm or an egg, then it may be inherited as an ‘endogenous’ retrovirus. In fact, endogenous retroviruses are extraordinarily common in mammalian DNA, constituting about 8% of human DNA. These endogenous retroviruses are mostly derived from ancient viruses that are now extinct. In this study, we recovered parts of two groups of extinct retroviruses, many strains of which became integrated into genomes of many nonhuman primates over the past few million years, but are absent from human DNA. We were able to generate infectious retroviruses by inserting a part of the extinct viruses into a modern retrovirus found in mice, and in so doing were able to functionally analyze properties of the extinct virus. Using a combination of these functional analyses, as well as sequence analysis, we obtained evidence that some rapidly evolving host defense molecules present in modern primates were able to inhibit the replication of these extinct viruses. Therefore, particular host defenses may have limited transmission of ancient retroviruses between species and perhaps contributed to their extinction.
PMCID: PMC2564838  PMID: 18927623
4.  Positive Selection of Iris, a Retroviral Envelope–Derived Host Gene in Drosophila melanogaster 
PLoS Genetics  2005;1(4):e44.
Eukaryotic genomes can usurp enzymatic functions encoded by mobile elements for their own use. A particularly interesting kind of acquisition involves the domestication of retroviral envelope genes, which confer infectious membrane-fusion ability to retroviruses. So far, these examples have been limited to vertebrate genomes, including primates where the domesticated envelope is under purifying selection to assist placental function. Here, we show that in Drosophila genomes, a previously unannotated gene (CG4715, renamed Iris) was domesticated from a novel, active Kanga lineage of insect retroviruses at least 25 million years ago, and has since been maintained as a host gene that is expressed in all adult tissues. Iris and the envelope genes from Kanga retroviruses are homologous to those found in insect baculoviruses and gypsy and roo insect retroviruses. Two separate envelope domestications from the Kanga and roo retroviruses have taken place, in fruit fly and mosquito genomes, respectively. Whereas retroviral envelopes are proteolytically cleaved into the ligand-interaction and membrane-fusion domains, Iris appears to lack this cleavage site. In the takahashii/suzukii species groups of Drosophila, we find that Iris has tandemly duplicated to give rise to two genes (Iris-A and Iris-B). Iris-B has significantly diverged from the Iris-A lineage, primarily because of the “invention” of an intron de novo in what was previously exonic sequence. Unlike domesticated retroviral envelope genes in mammals, we find that Iris has been subject to strong positive selection between Drosophila species. The rapid, adaptive evolution of Iris is sufficient to unambiguously distinguish the phylogenies of three closely related sibling species of Drosophila (D. simulans, D. sechellia, and D. mauritiana), a discriminative power previously described only for a putative “speciation gene.” Iris represents the first instance of a retroviral envelope–derived host gene outside vertebrates. It is also the first example of a retroviral envelope gene that has been found to be subject to positive selection following its domestication. The unusual selective pressures acting on Iris suggest that it is an active participant in an ongoing genetic conflict. We propose a model in which Iris has “switched sides,” having been recruited by host genomes to combat baculoviruses and retroviruses, which employ homologous envelope genes to mediate infection.
Mobile genetic elements have made homes within eukaryotic (host) genomes for hundreds of millions of years. These include retroviruses that integrate into host genomes as an essential step in their life cycle. While most such integration events are likely to be either deleterious or of little consequence to the host, on rare occasions host genomes can preserve and exploit capabilities of mobile elements for their own function. Especially intriguing are instances where host genomes have chosen to retain the envelope genes of retroviruses; the same envelope genes are responsible for conferring infectious ability to retroviruses. Primates and rodent genomes each have domesticated retroviral envelope genes (called “syncytin” genes) for important roles in placental function.
Now, Harmit Malik and colleagues show that a similar, ancient domestication event has taken place within the fruit fly Drosophila melanogaster. They identify a gene, Iris, which was acquired from an envelope gene of insect retroviruses, and has been maintained as a host gene for more than 25 million years. Unexpectedly, the authors find that Iris continues to evolve rapidly whereas previous studies have shown that mammalian syncytin genes do not. They suggest a model in which the Iris gene has “switched sides,” from its original role in causing infections to its current role in preventing them.
PMCID: PMC1262188  PMID: 16244705
5.  The ancient Virus World and evolution of cells 
Biology Direct  2006;1:29.
Recent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones.
Several genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of precellular evolution under which the primordial gene pool dwelled in a network of inorganic compartments. Somewhat paradoxically, under this scenario, we surmise that selfish genetic elements ancestral to viruses evolved prior to typical cells, to become intracellular parasites once bacteria and archaea arrived at the scene. Selection against excessively aggressive parasites that would kill off the host ensembles of genetic elements would lead to early evolution of temperate virus-like agents and primitive defense mechanisms, possibly, based on the RNA interference principle. The emergence of the eukaryotic cell is construed as the second melting pot of virus evolution from which the major groups of eukaryotic viruses originated as a result of extensive recombination of genes from various bacteriophages, archaeal viruses, plasmids, and the evolving eukaryotic genomes. Again, this vision is predicated on a specific model of the emergence of eukaryotic cell under which archaeo-bacterial symbiosis was the starting point of eukaryogenesis, a scenario that appears to be best compatible with the data.
The existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life's evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life's history.
W. Ford Doolittle, J. Peter Gogarten, and Arcady Mushegian.
PMCID: PMC1594570  PMID: 16984643
6.  Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases 
Over 40% of the human genome is recognizable as having been derived from ancient retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate, with an additional few percent classified as DNA transposable elements. Endogenous retroviruses are long terminal repeat (LTR)-type retroelements that account for ~8% of human genomic DNA. Non-LTR members are present at extremely high copy numbers, with ~17% of the human genome consisting of long interspersed nuclear elements (LINEs). These LINEs modify vertebrate genomes not only through insertions, but also by the indirect replication of non-autonomous retrotransposons, such as short interspersed nuclear elements. As expected, vertebrate intrinsic immunity has evolved to support a balance between retroelement insertions that confer beneficial genetic diversity and those that cause deleterious gene disruptions. The mammalian cytidine deaminases encoded by the APOBEC3 genes can restrict a broad number of exogenous pathogens, such as exogenous retroviruses, and the mobility of endogenous retroelements. Furthermore, APOBEC1 from a variety of mammalian species, which mediates the cytidine (C) to uridine (U) deamination of apolipoprotein B (apoB) mRNA, a protein involved in lipid transport, also plays a role in controlling mobile elements. These mammalian apoB mRNA-editing, catalytic polypeptide (APOBEC) cytidine deaminases, which can bind to single-stranded DNA (ssDNA) as well as RNA, are able to insert mutations into ssDNA and/or RNA as a result of their ability to deaminate C to U. While these APOBEC cytidine deaminases with DNA mutagenic activity can be deleterious to cells, their biological modifications, such as protein–protein interactions and subcellular localization, in addition to their ability to bind to RNA, appear to have conferred a role for APOBECs as a cellular defense system against retroviruses and retroelements. In support of this notion, the expansion of the single APOBEC3 gene in mice to the seven APOBEC3 genes found in primates apparently correlates with the significant enhancement of the restriction of endogenous retroelements seen in primates, including humans. This review discusses the current understanding of the mechanism of action of APOBEC cytidine deaminases and attempts to summarize their roles in controlling retrotransposons.
PMCID: PMC3576619  PMID: 23431045
APOBEC1; APOBEC3; AID; retrovirus; HIV-1; LINE-1; retroelements; endogenous retrovirus
7.  INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites 
Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra- from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (4′,6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the “gold standard” microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites.
Author Summary
Research on intracellular parasites require using non-invasive technologies to follow up parasite proliferation inside their natural host cells by staying in the more physiological conditions as possible. High Content Screening (HCS) technology has recently emerged as a powerful image-based approach to screen new anti-parasitic compounds or to test parasite susceptibility to existing drugs in vitro. Nevertheless, such equipments will remain poorly accessible for most of academic and clinical diagnostic laboratories that mostly use more affordable, but laborious, microscopic counting procedures. The current work proposes new image-based, open-source software which provides a fast and accurate solution for investigating intracellular parasite quantification. Through an easy-to-use interface, cells' and parasites' information are dug out from DNA fluorescent images, and host cells' boundaries are extracted from corresponding phase contrast image set. Parasites are then reassigned to their related cells and intra/extracellular parasites are discriminated for each cell. The software further automatically calculates all data required for most of experimental infection studies. INsPECT software is proposed as a free substitute or complement to the available quantification methods for measuring Leishmania infection level in vitro. It may be enlarged, however, to different intracellular trypanosomatids or unrelated parasites such as T. gondii.
PMCID: PMC4022486  PMID: 24831235
8.  Intrinsic restriction activity by AID/APOBEC family of enzymes against the mobility of retroelements 
Mobile Genetic Elements  2011;1(3):197-202.
A large portion of the mammalian genome is derived from ancient transposable elements. Retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate (retrotransposition), constitute a majority of these mobile genetic elements. Endogenous retroviruses are LTR-type retroelements accounting for around 8% of human or murine genomic DNA. Non-LTR members are present in extremely high copy numbers; with LINE-1 contributing to nearly 40% of human and murine genomes. These LINE-1 elements modify mammalian genomes not only through insertions, but also by indirect replication of nonautonomous retrotransposons such as SINEs. As expected, cellular machineries of vertebrate's innate immunity have evolved to support a balance between retroelement insertions that cause deleterious gene disruptions and those that confer beneficial genetic diversity. The ability of APOBEC3 cytidine deaminases targeting DNA to restrict a broad number of retroviruses and retro-elements is now well established. More recently, the RNA editing family member APOBEC1, a protein involved in lipid transport, has also been shown to be involved in keeping mobile elements under control. This review discusses current understanding of the mechanism of action of the AID/APOBEC family, and their role in controlling the retrotransposition of endogenous retroelements.
PMCID: PMC3312301  PMID: 22479686
APOBEC1; APOBEC3; AID; LINE-1; retroelements; endogenous retrovirus; hypermutation; RNA editing; DNA deamination
9.  Relationships of gag-pol diversity between Ty3/Gypsy and Retroviridae LTR retroelements and the three kings hypothesis 
The origin of vertebrate retroviruses (Retroviridae) is yet to be thoroughly investigated, but due to their similarity and identical gag-pol (and env) genome structure, it is accepted that they evolve from Ty3/Gypsy LTR retroelements the retrotransposons and retroviruses of plants, fungi and animals. These 2 groups of LTR retroelements code for 3 proteins rarely studied due to the high variability – gag polyprotein, protease and GPY/F module. In relation to 3 previously proposed Retroviridae classes I, II and II, investigation of the above proteins conclusively uncovers important insights regarding the ancient history of Ty3/Gypsy and Retroviridae LTR retroelements.
We performed a comprehensive study of 120 non-redundant Ty3/Gypsy and Retroviridae LTR retroelements. Phylogenetic reconstruction inferred based on the concatenated analysis of the gag and pol polyproteins shows a robust phylogenetic signal regarding the clustering of OTUs. Evaluation of gag and pol polyproteins separately yields discordant information. While pol signal supports the traditional perspective (2 monophyletic groups), gag polyprotein describes an alternative scenario where each Retroviridae class can be distantly related with one or more Ty3/Gypsy lineages. We investigated more in depth this evidence through comparative analyses performed based on the gag polyprotein, the protease and the GPY/F module. Our results indicate that contrary to the traditional monophyletic view of the origin of vertebrate retroviruses, the Retroviridae class I is a molecular fossil, preserving features that were probably predominant among Ty3/Gypsy ancestors predating the split of plants, fungi and animals. In contrast, classes II and III maintain other phenotypes that emerged more recently during Ty3/Gypsy evolution.
The 3 Retroviridae classes I, II and III exhibit phenotypic differences that delineate a network never before reported between Ty3/Gypsy and Retroviridae LTR retroelements. This new scenario reveals how the diversity of vertebrate retroviruses is polyphyletically recurrent into the Ty3/Gypsy evolution, i.e. older than previously thought. The simplest hypothesis to explain this finding is that classes I, II and III trace back to at least 3 Ty3/Gypsy ancestors that emerged at different evolutionary times prior to protostomes-deuterostomes divergence. We have called this "the three kings hypothesis" concerning the origin of vertebrate retroviruses.
PMCID: PMC2577118  PMID: 18842133
10.  Trypanosoma cruzi IIc: Phylogenetic and Phylogeographic Insights from Sequence and Microsatellite Analysis and Potential Impact on Emergent Chagas Disease 
Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.
Author Summary
Trypanosoma cruzi, the etiological agent of Chagas disease, infects over 10 million people in Latin America. Six major genetic lineages of the parasite have been identified with differential geographic distributions, ecological associations and epidemiological importance. With the advent of the T. cruzi genome sequence, it is possible to examine the micro-epidemiology of T. cruzi using high resolution genetic markers that assess diversity within these major types. Here we examine the genetic diversity of TcIIc, a poorly understood T. cruzi genetic lineage found predominantly among wild cycles of parasite transmission infecting terrestrial mammals and triatomine vectors, but also a potentially important emergent human disease agent. Amongst a number of findings, we show that TcIIc genetic diversity is comparable to other ancient T. cruzi lineages, highly spatially structured, and that a stringent co-evolutionary relationship with its principal reservoir host can be ruled out. Additionally, TcIIc is one of the two parents of hybrid lineages TcIId and TcIIe, which cause most of the Chagas disease that occurs in the Southern Cone of South America. The system we have developed will help to clarify the ecological circumstances around the emergence of these epidemiologically important hybrids, and perhaps help predict similar events in the future.
PMCID: PMC2727949  PMID: 19721699
11.  An Endogenous Foamy-like Viral Element in the Coelacanth Genome 
PLoS Pathogens  2012;8(6):e1002790.
Little is known about the origin and long-term evolutionary mode of retroviruses. Retroviruses can integrate into their hosts' genomes, providing a molecular fossil record for studying their deep history. Here we report the discovery of an endogenous foamy virus-like element, which we designate ‘coelacanth endogenous foamy-like virus’ (CoeEFV), within the genome of the coelacanth (Latimeria chalumnae). Phylogenetic analyses place CoeEFV basal to all known foamy viruses, strongly suggesting an ancient ocean origin of this major retroviral lineage, which had previously been known to infect only land mammals. The discovery of CoeEFV reveals the presence of foamy-like viruses in species outside the Mammalia. We show that foamy-like viruses have likely codiverged with their vertebrate hosts for more than 407 million years and underwent an evolutionary transition from water to land with their vertebrate hosts. These findings suggest an ancient marine origin of retroviruses and have important implications in understanding foamy virus biology.
Author Summary
The deep history of retroviruses is still obscure. Retroviruses can leave integrated copies within their hosts' genomes, providing a fossil record for studying their long-term evolution. Endogenous forms of foamy viruses, complex retroviruses known to infect only mammalian species, appear to be extremely rare, so far found only in sloths and the aye-aye. Here, we report the discovery of endogenous foamy virus-like insertions within the genome of a so-called ‘living fossil’, the coelacanth (Latimeria chalumnae). We provide evidence suggesting that foamy viruses and their hosts share a coevolutionary history of more than 407 million years, and that foamy viruses accompanied their vertebrate hosts on the evolutionary transition from water to land. These findings indicate that the retroviruses originated in the primeval ocean millions of years ago.
PMCID: PMC3386198  PMID: 22761578
12.  RNAs from genetically distinct retroviruses can copackage and exchange genetic information in vivo. 
Journal of Virology  1997;71(8):6237-6242.
Sequence analysis suggests that ancient recombination events may have occurred between genetically distinct retroviruses. An experimental system was utilized to explore the genetic interaction between different viruses. Moloney murine sarcoma virus and spleen necrosis virus are type C retroviruses that belong to different subgenera. With vectors containing packaging signals from these two viruses, DNA proviruses containing genetic information from both RNAs can be generated. This is the first experimental evidence to indicate that RNA from different retroviruses can copackage and exchange genetic information.
PMCID: PMC191891  PMID: 9223525
13.  T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection 
PLoS Pathogens  2007;3(11):e165.
Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.
Author Summary
The human genome contains a number of remnants or fossils of ancient viral infections referred to as human endogenous retroviruses (HERV). Like fossils, these HERV are considered to be dead or inert in most cases. However, we demonstrate that T cells in the human immune system respond to HERV when a person is infected with the human immunodeficiency virus (HIV). The T cells responding to HERV share characteristics with T cells that effectively control cytomegalovirus, a common chronic viral infection. T cells responding to HERV can also kill target cells carrying HERV protein. For some HIV-positive people, the strength of their response against HERV is related to having a lower HIV viral load. This study has important implications for new directions in HIV vaccine research. One of the key obstacles to creating an effective HIV vaccine is overcoming the ability of some of the viral variants produced when HIV replicates to evade the immune responses that the body mounts to control infections. If T cells that recognize HERV can stably target HIV-infected cells, they could be an important factor in controlling HIV infection.
PMCID: PMC2065876  PMID: 17997601
14.  Reconstitution of an Infectious Human Endogenous Retrovirus 
PLoS Pathogens  2007;3(1):e10.
The human genome represents a fossil record of ancient retroviruses that once replicated in the ancestors of contemporary humans. Indeed, approximately 8% of human DNA is composed of sequences that are recognizably retroviral. Despite occasional reports associating human endogenous retrovirus (HERV) expression with human disease, almost all HERV genomes contain obviously inactivating mutations, and none are thought to be capable of replication. Nonetheless, one family of HERVs, namely HERV-K(HML-2), may have replicated in human ancestors less than 1 million years ago. By deriving a consensus sequence, we reconstructed a proviral clone (HERV-KCON) that likely resembles the progenitor of HERV-K(HML-2) variants that entered the human genome within the last few million years. We show that HERV-KCON Gag and protease proteins mediate efficient assembly and processing into retrovirus-like particles. Moreover, reporter genes inserted into the HERV-KCON genome and packaged into HERV-K particles are capable of infectious transfer and stable integration in a manner that requires reverse transcription. Additionally, we show that HERV-KCON Env is capable of pseudotyping HIV-1 particles and mediating entry into human and nonhuman cell lines. Furthermore, we show that HERV-KCON is resistant to inhibition by the human retrovirus restriction factors tripartite motif 5α and apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 3G but is inhibited by APOBEC 3F. Overall, the resurrection of this extinct infectious agent in a functional form from molecular fossils should enable studies of the molecular virology and pathogenic potential of this ancient human retrovirus.
Author Summary
Retrovirus genomes integrate into the genomes of host cells. If the target cells of a particular retrovirus include germ-line cells, e.g., sperm or egg cells, then retroviral genomes can be inherited like cellular genes. So-called “endogenous” retroviruses have accumulated throughout evolution in the genomes of many organisms, including humans. While all known endogenous retroviruses of modern humans are unable to replicate as retroviruses, the human genome represents a fossil record of ancient retroviruses that once infected our ancestors. In this study, a collection of “dead” endogenous retroviral genomes in modern human DNA was used to deduce the approximate sequence of an ancestral retrovirus, human endogenous retrovirus (HERV)-K, that is now thought to be extinct. A pseudo-ancestral HERV-K DNA sequence was synthesized and used to produce viral proteins and RNA that could reconstitute the HERV-K replication cycle. Thus, the replication and biology of a once-extinct retrovirus can now be studied in the laboratory. Interestingly, reconstituted HERV-K replication experiments, and comparison of the reconstituted HERV-K DNA sequence with the dead HERV-Ks in modern human DNA, suggests that HERV-K may have been extinguished in humans in part by host defenses that induce mutation of retroviral DNA and that the reconstitution of the pseudo-ancestral HERV-K reversed these changes.
PMCID: PMC1781480  PMID: 17257061
15.  Multipotent Genetic Suppression of Retrotransposon-Induced Mutations by Nxf1 through Fine-Tuning of Alternative Splicing 
PLoS Genetics  2009;5(5):e1000484.
Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1CAST suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including Ap3d1mh2J, a model for Hermansky-Pudlak syndrome, and Atcayhes, an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes ∼two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1CAST alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.
Author Summary
Retroviruses and transposable elements are molecular parasites that integrate into the host genome and require host cell machinery for gene expression, replication and dissemination. Integrating elements can alter the expression of nearby host genes through both transcriptional and post-transcriptional mechanisms. Components of the host cell machinery that can adapt to favor genetic programs of the host cell over those of the parasite may afford one level of innate immunity. In laboratory mice, endogenous retroviruses are virus-derived mobile elements that account for many spontaneous mutations. A frequent class involves retrotransposition into introns of genes in the transcriptional sense orientation, which alters host gene pre-mRNA splicing. Here we show that for the intracisternal A particle (IAP) family of endogenous retroviruses, an allele of the canonical mRNA export factor Nxf1 found in wild Asiatic mice (Mus musculus castaneus) suppresses most insertions of this class (six of seven tested). To our knowledge, these results make Nxf1 the most broadly interacting modifier gene yet documented in this well-studied species. These results have significant implications for manipulating gene expression in mouse models of disease, the role of Nxf1 in pre-mRNA processing and in the dynamic range for therapeutic intervention in Cayman ataxia.
PMCID: PMC2674570  PMID: 19436707
16.  Function of a retrotransposon nucleocapsid protein 
RNA Biology  2010;7(6):642-654.
Long terminal repeat (LTR) retrotransposons are not only the ancient predecessors of retroviruses, but they constitute significant fractions of the genomes of many eukaryotic species. Studies of their structure and function are motivated by opportunities to gain insight into common functions of retroviruses and retrotransposons, diverse mechanisms of intracellular genomic mobility and host factors that diminish or enhance retrotransposition. This review focuses on the nucleocapsid (NC) protein of a Saccharomyces cerevisiae LTR retrotransposon, the metavirus, Ty3. Retrovirus NC promotes genomic (g)RNA dimerization and packaging, tRNA primer annealing, reverse transcription strand transfers, and host protein interactions with gRNA. Studies of Ty3 NC have revealed key roles for Ty3 NC in formation of retroelement assembly sites (retrosomes), and in chaperoning primer tRNA to both dimerize and circularize Ty3 gRNA. We speculate that Ty3 NC, together with P-body and stress-granule proteins, plays a role in transitioning Ty3 RNA from translation template to gRNA, and that interactions between the acidic spacer domain of Ty3 Gag3 and the adjacent basic NC domain control condensation of the virus-like particle.
PMCID: PMC3073325  PMID: 21189452
nucleocapsid; retroelement; retrotransposon; retrovirus; Ty; reverse transcription; assembly
17.  Landforms predict phylogenetic structure on one of the world's most ancient surfaces 
The iconic Pilbara in northwestern Australia is an ancient geological and biophysical region that is an important zone of biodiversity, endemism and refugia. It also is overlain by some of the oldest erosion surfaces on Earth, but very little is known about the patterns of biotic diversity within the Pilbara or how they relate to the landscape. We combined phylogenetic and spatial-autocorrelation genetic analyses of mitochondrial DNA data on populations of the gekkotan lizard Lucasium stenodactylum within the Pilbara with geological, distributional and habitat data to test the hypothesis that ancient surface geology predicts current clade-habitat associations in saxicoline animals.
This is the first detailed phylogenetic examination of a vertebrate organism across the Pilbara region. Our phylogeny provides strong support for a deep and ancient phylogenetic split within L. stenodactylum that distinguishes populations within the Pilbara region from those outside the Pilbara. Within the Pilbara region itself, our phylogeny has identified five major clades whose distribution closely matches different surface geologies of this ancient landscape. Each clade shows strong affinities with particular terrain types and topographic regions, which are directly related to different geological bedrock.
Together our phylogenetic, distributional, geological and habitat data provide a clear example of ecological diversification across an ancient and heterogeneous landscape. Our favoured hypothesis is that ancestors of the Pilbara lineages radiated into the region at the onset of aridity in Australia approximately 5 mya and locally adapted to the various ancient and highly stable terrain types and the micro-habitats derived from them. In terms of specimen recovery and analysis, we are only beginning to reconstruct the biotic history of this ancient landscape. Our results show the geological history and the habitats derived from them will form an important part of this emerging story.
PMCID: PMC2397392  PMID: 18485245
18.  Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection 
PLoS Pathogens  2009;5(5):e1000410.
Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is “hidden” at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed.
Author Summary
The arrival of the Trypanosoma cruzi online genome now provides vital information for the study of Chagas disease. Using this resource, we identified and developed a genome-scale panel of rapidly evolving microsatellite markers that can be used to unravel the micro-epidemiology of this parasite. We then tested these against a panel of isolates belonging to the most widely occurring and ancient major lineage, T. cruzi I (TcI). Our study includes samples from across the geographical distribution of this lineage, including isolates from wild vectors, domestic vectors, as well as wild mammalian reservoirs and human hosts. This is the first time T. cruzi has been subjected to such high-resolution population genetic analysis. Our study shows that important epidemiological information lies at the intra-lineage level, especially when wild and domestic populations of parasite are compared. Crucially, in Venezuela, where Chagas disease may be resurgent despite decades of control effort, genotypes of parasites found in the wild are rarely represented in humans, despite evidence that infected wild vectors do invade houses. In this manuscript, we examine the epidemiological implications of this finding and others, and suggest how the approach we have developed can now be used to investigate the true nature of parasite transmission at Chagas disease foci throughout the Americas.
PMCID: PMC2669174  PMID: 19412340
19.  Association of Endogenous Retroviruses and Long Terminal Repeats with Human Disorders 
Frontiers in Oncology  2013;3:234.
Since the human genome sequences became available in 2001, our knowledge about the human transposable elements which comprise ∼40% of the total nucleotides has been expanding. Non-long terminal repeat (non-LTR) retrotransposons are actively transposing in the present-day human genome, and have been found to cause ∼100 identified clinical cases of varied disorders. In contrast, almost all of the human endogenous retroviruses (HERVs) originating from ancient infectious retroviruses lost their infectivity and transposing activity at various times before the human-chimpanzee speciation (∼6 million years ago), and no known HERV is presently infectious. Insertion of HERVs and mammalian apparent LTR retrotransposons (MaLRs) into the chromosomal DNA influenced a number of host genes in various modes during human evolution. Apart from the aspect of genome evolution, HERVs and solitary LTRs being suppressed in normal biological processes can potentially act as extra transcriptional apparatuses of cellular genes by re-activation in individuals. There has been a reasonable prediction that aberrant LTR activation could trigger malignant disorders and autoimmune responses if epigenetic changes including DNA hypomethylation occur in somatic cells. Evidence supporting this hypothesis has begun to emerge only recently: a MaLR family LTR activation in the pathogenesis of Hodgkin’s lymphoma and a HERV-E antigen expression in an anti-renal cell carcinoma immune response. This mini review addresses the impacts of the remnant-form LTR retrotransposons on human pathogenesis.
PMCID: PMC3769647  PMID: 24062987
human endogenous retrovirus; HERV; long terminal repeat; LTR; cancer; retrotransposon; autoimmune
20.  Cell Division in Apicomplexan Parasites Is Organized by a Homolog of the Striated Rootlet Fiber of Algal Flagella 
PLoS Biology  2012;10(12):e1001444.
Apicomplexan parasites undergo cell division using an evolutionarily conserved mechanism first described in the positioning and assembly of flagella in algae.
Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in Toxoplasma gondii demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.
Author Summary
Malaria, toxoplasmosis, and related diseases are caused by infection with unicellular parasites called Apicomplexa. Their name refers to the elaborate invasion machinery that occupies the apical end of the parasite cell. This apparatus allows the parasite to force its way into the cells of its host, and to deliver factors that will manipulate host cell structure, gene expression, and metabolism. Once in the host cell the parasite will begin to grow. The parasite replicates its genome and organelles numerous times and then loads these various elements into numerous daughter cells that will further spread the infection. Here we report a fiber that coordinates the daughter cell budding process. The fiber links the centrosome, which controls the mitotic spindle, and the genome with the microtubule organizing center of the budding daughter. Parasite mutants lacking the proteins that build the fiber fail to form daughter cells at the earliest step. The fiber and its components are remarkably similar to fibers that coordinate flagella in algae. While Apicomplexa are not flagellated (with the exception of certain gamete stages) they evolved from flagellated algae. We propose that elements of the invasion apparatus evolved from the flagellum or flagellum associated structures.
PMCID: PMC3519896  PMID: 23239939
21.  Improved Integration Time Estimation of Endogenous Retroviruses with Phylogenetic Data 
PLoS ONE  2011;6(3):e14745.
Endogenous retroviruses (ERVs) are genetic fossils of ancient retroviral integrations that remain in the genome of many organisms. Most loci are rendered non-functional by mutations, but several intact retroviral genes are known in mammalian genomes. Some have been adopted by the host species, while the beneficial roles of others remain unclear. Besides the obvious possible immunogenic impact from transcribing intact viral genes, endogenous retroviruses have also become an interesting and useful tool to study phylogenetic relationships. The determination of the integration time of these viruses has been based upon the assumption that both 5′ and 3′ Long Terminal Repeats (LTRs) sequences are identical at the time of integration, but evolve separately afterwards. Similar approaches have been using either a constant evolutionary rate or a range of rates for these viral loci, and only single species data. Here we show the advantages of using different approaches.
We show that there are strong advantages in using multiple species data and state-of-the-art phylogenetic analysis. We incorporate both simple phylogenetic information and Monte Carlo Markov Chain (MCMC) methods to date the integrations of these viruses based on a relaxed molecular clock approach over a Bayesian phylogeny model and applied them to several selected ERV sequences in primates. These methods treat each ERV locus as having a distinct evolutionary rate for each LTR, and make use of consensual speciation time intervals between primates to calibrate the relaxed molecular clocks.
The use of a fixed rate produces results that vary considerably with ERV family and the actual evolutionary rate of the sequence, and should be avoided whenever multi-species phylogenetic data are available. For genome-wide studies, the simple phylogenetic approach constitutes a better alternative, while still being computationally feasible.
PMCID: PMC3048862  PMID: 21394200
22.  Phylogenetic distribution of the novel avian endogenous provirus family EAV-0. 
Journal of Virology  1990;64(10):4640-4653.
A new family of related endogenous proviruses, existing at 50 to 100 copies per haploid genome and distinguishable by remarkably short long terminal repeats, has been described for domestic chickens (Gallus gallus subsp domesticus). In this communication, by using Southern blot analysis and probes derived from both internal viral sequences and locus-specific, cellular flanking sequences, we studied the genetic distribution of this family of moderately repetitive avian endogenous retroviruses within the genomes of four Gallus species. Eight inbred lines of domestic chickens, the evolutionary progenitor to the domestic chicken (red jungle fowl), and two more distantly related species (grey and green jungle fowl) were studied. All Gallus species harbored this class of elements, although the different lines of domestic chickens and different species of jungle fowl bore distinguishable complements of the proviral loci. Jungle fowl appeared to have fewer copies than domestic chickens. For three randomly isolated proviral loci, domestic chickens (G. gallus subsp. domesticus) and red jungle fowl (G. gallus subsp. gallus) showed only a proviral state, whereas the most primitive and divergent of the jungle fowl, the green jungle fowl (G. varius), consistently demonstrated only preintegration states or disparate alleles. The presence of this family in all Gallus species and of related sequences in other genera suggests that a primordial founding integration event occurred prior to the evolutionary separation of Gallus species and possibly related genera. Additionally, at least one proviral locus has been acquired subsequent to speciation, indicating that this family was actively infectious after the primary founding event. This conserved, repetitive proviral family appears to represent the vestigial remnant of an avian retrovirus class related to and evolutionarily more ancient than the Rous-associated virus-0 family of avian endogenous retroviruses.
PMCID: PMC247948  PMID: 2398526
23.  Molecular characterization of the evolution of phagosomes 
First large-scale comparative proteomics/phosphoproteomics study characterizing some of the key steps that contributed to the remodeling of phagosomes that occurred during evolution. Comparison of profiling analyses of isolated phagosomes from three distant organisms (Dictyostelium, Drosophila, and mouse) revealed a protein core that defines a potential ‘ancient' phagosome and a set of 50 proteins that emerged while adaptive immunity was already well established.Gene duplication events of mouse phagosome paralogs occurred mostly in Bilateria and Euteleostomi, coinciding with the emergence of innate and adaptive immunity, and thus, provided the functional innovations needed for the establishment of these two crucial evolutionary steps of the immune system.Phosphoproteomics of isolated phagosomes from the same three distant species indicate that the phagosome phosphoproteome has been extensively modified during evolution. Still, some phosphosites have been maintained for >1.2 billion years, and thus, highlight their particular significance in the regulation of key phagosomal functions.
Phagocytosis is the process by which multiple cell types internalize large particulate material from the external milieu. The functional properties of phagosomes are acquired through a complex maturation process, referred to as phagolysosome biogenesis. This pathway involves a series of rapid interactions with organelles of the endocytic apparatus, enabling the gradual transformation of newly formed phagosomes into phagolysosomes in which proteolytic degradation occurs. The degradative environment encountered in the phagosome lumen has enabled the use of phagocytosis as a predation mechanism for feeding (phagotrophy) in amoeba, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in jawed vertebrates (fish), initiate a sustained immune response.
High-throughput proteomics profiling of isolated phagosomes has been tremendously helpful for the molecular comprehension of this organelle. This approach is achieved by feeding low buoyancy latex beads to phagocytic cells, enabling the subsequent isolation of latex bead-containing phagosomes, away from all the other cell organelles, by a single-isopicnic centrifugation in sucrose gradient. In order to characterize some of the key steps that contributed to the remodeling of phagosomes during evolution, we isolated this organelle from three distant organisms: the amoeba Dictyostelium discoideum, the fruit fly Drosophila melanogaster, and mouse (Mus musculus) that use phagocytosis for different purposes, and performed detailed proteomics and phosphoproteomics analyses with unparallel protein coverage for this organelle (two- to four-fold enhancements in identified proteins).
In order to establish the origin of the mouse phagosome proteome, we performed comparative analyses among 39 taxa including plants/algea, unicellular organisms, fungi, and more complex animal multicellular organisms. These genomic comparisons indicated that a large proportion of the mouse phagosome proteome is of ancient origin (73.1% of the proteome is conserved in eukaryotic organisms) (Figure 2A). This stresses the fact that phagocytosis is a very ancient process, as shown by its possible involvement in the emergence of eukaryotic cells (eukaryogenesis). Indeed, we identified close to 300 phagosome mouse proteins also present on Drosophila and Dictyostelium phagosomes, defining a potential ‘ancient' core of proteins from which the immune functions of phagosomes likely evolved. Around 16.7% of the mouse phagosome proteins appeared in organisms that use phagocytosis for innate immunity (Bilateria to Chordata), whereas 10.2% appeared in Euteleostomi or Tetrapoda where phagosomes have an important function in linking the killing of microorganisms with the development of a specific sustained immune response following antigen recognition. The phagosome is made of molecules taken from a variety of sources within the cell, including the cytoplasm, the cytoskeleton and membrane organelles. Despite the evolution and diversification of these various cellular systems, the mammalian phagosome proteome is made preferentially of ancient proteins (Figure 2B). Comparison of functional annotation during evolution highlighted the emergence of specific phagosomal functions at various steps during evolution (Figure 2C). Some of these proteins and their point of origin during evolution are highlighted in Figure 2D. Strikingly, we identified in Tetrapods a set of 50 proteins that arose while adaptive immunity was already well established in teleosts (fish), indicating that the phagocytic system is still evolving.
Our study highlights the fact that the functional properties of phagosomes emerged by the remodeling of ancient molecules, the addition of novel components, and the duplication of existing proteins (paralogs) leading to the formation of molecular machines of mixed origin. Gene duplication is a process that contributed continuously to the complexification of the mouse proteome during evolution. In sharp contrast, paralog analysis indicated that the phagosome proteome was mainly reorganized through two periods of gene duplication, in Bilateria and Euteleostomi, coinciding with the emergence of adaptive immunity (in jawed fish), and innate immunity (at the split between Metazoa and Bilateria). These results strongly suggest that selective constraints may have favored the maintenance of phagosome paralogs to ensure the establishment of novel functions associated with this organelle at these two crucial evolutionary steps of the immune system.
The emergence of genes associated to the MHC locus in mammals that appeared originally in the genome of jawed fishes, contributed to the development of complex molecular mechanisms linking innate (our immune system that defends the host from infection in a non-specific manner) and adaptive immunity (the part of the immune system triggered specifically after antigen recognition). Several of the genes of this locus encode proteins known to have important functions in antigen presentation, such as subunits of the immunoproteasome (LMP2 and LMP7), MHC class I and class II molecules, as well as tapasin and the transporter associated with antigen processing (TAP1 and TAP2), involved in the transport and loading of peptides on MHC class I molecules (Figure 6). In addition to their ability to present peptides on MHC class II molecules, phagosomes of vertebrates have been shown to be competent for the presentation of exogenous peptides on MHC class I molecules, a process referred to as cross-presentation. From a functional point of view, the involvement of phagosomes in antigen cross-presentation is the outcome of the successful integration of a wide range of multimolecular components that emerged throughout evolution (Figure 6). The trimming of exogenous proteins into small peptides that can be loaded on MHC class I molecules is inherited from the phagotrophic properties of unicellular organisms, where internalized bacteria are degraded into basic molecules and used as a source of nutrients. Ancient processes have therefore been co-opted (the use of an existing biological structure or feature for a new function) for new functionalities. A summarizing model of the various steps that enabled phagosome antigen presentation is presented in Figure 6. This model highlights the fact that although antigen presentation is unique to evolutionary recent phagosomes (starting in jawed fishes about 450 million years ago), it uses and integrates molecular machines composed of proteins that emerged throughout evolution.
In summary, we present here the first large-scale comparative proteomics/phosphoproteomics study characterizing some of the key evolutionary steps that contributed to the remodeling of phagosomes during evolution. Functional properties of this organelle emerged by the remodeling of ancient molecules, the addition of novel components, the extensive adaption of protein phosphorylation sites and the duplication of existing proteins leading to the formation of molecular machines of mixed origin.
Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an ‘ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.
PMCID: PMC2990642  PMID: 20959821
evolution; immunity; phosphoproteomics; phylogeny; proteomics
24.  On the Age of Leprosy 
Leprosy is a chronic infection of the skin and nerves caused by Mycobacterium leprae and the newly discovered Mycobacterium lepromatosis. Human leprosy has been documented for millennia in ancient cultures. Recent genomic studies of worldwide M. leprae strains have further traced it along global human dispersals during the past ∼100,000 years. Because leprosy bacilli are strictly intracellular, we wonder how long humans have been affected by this disease-causing parasite. Based on recently published data on M. leprae genomes, M. lepromatosis discovery, leprosy bacilli evolution, and human evolution, it is most likely that the leprosy bacilli started parasitic evolution in humans or early hominids millions of years ago. This makes leprosy the oldest human-specific infection. The unique adaptive evolution has likely molded the indolent growth and evasion from human immune defense that may explain leprosy pathogenesis. Accordingly, leprosy can be viewed as a natural consequence of a long parasitism. The burden of leprosy may have affected minor selection on human genetic polymorphisms.
PMCID: PMC3923669  PMID: 24551248
25.  Recent, Independent and Anthropogenic Origins of Trypanosoma cruzi Hybrids 
The single celled eukaryote Trypanosoma cruzi, a parasite transmitted by numerous species of triatomine bug in the Americas, causes Chagas disease in humans. T. cruzi generally reproduces asexually and appears to have a clonal population structure. However, two of the six major circulating genetic lineages, TcV and TcVI, are TcII-TcIII inter-lineage hybrids that are frequently isolated from humans in regions where chronic Chagas disease is particularly severe. Nevertheless, a prevalent view is that hybridisation events in T. cruzi were evolutionarily ancient and that active recombination is of little epidemiological importance. We analysed genotypes of hybrid and non-hybrid T. cruzi strains for markers representing three distinct evolutionary rates: nuclear GPI sequences (n = 88), mitochondrial COII-ND1 sequences (n = 107) and 28 polymorphic microsatellite loci (n = 35). Using Maximum Likelihood and Bayesian phylogenetic approaches we dated key evolutionary events in the T. cruzi clade including the emergence of hybrid lineages TcV and TcVI, which we estimated to have occurred within the last 60,000 years. We also found evidence for recent genetic exchange between TcIII and TcIV and between TcI and TcIV. These findings show that evolution of novel recombinants remains a potential epidemiological risk. The clearly distinguishable microsatellite genotypes of TcV and TcVI were highly heterozygous and displayed minimal intra-lineage diversity indicative of even earlier origins than sequence-based estimates. Natural hybrid genotypes resembled typical meiotic F1 progeny, however, evidence for mitochondrial introgression, absence of haploid forms and previous experimental crosses indicate that sexual reproduction in T. cruzi may involve alternatives to canonical meiosis. Overall, the data support two independent hybridisation events between TcII and TcIII and a recent, rapid spread of the hybrid progeny in domestic transmission cycles concomitant with, or as a result of, disruption of natural transmission cycles by human activities.
Author Summary
Trypanosoma cruzi is a parasite that causes Chagas disease in humans, an often fatal condition affecting at least 8 million people. The clinical outcome of Chagas disease is variable, which is probably partially attributable to genetic differences between T. cruzi strains. Differences between T. cruzi strains can arise by gradual accumulation of mutations in independent lineages (clonal reproduction) or by recombination between strains, which generates new combinations of existing alleles (sexual reproduction). Although sex has been observed experimentally, and epidemiologically important T. cruzi subgroups (TcV TcVI) originated via hybridisation events, active recombination is considered to be extremely rare. Our aim was to determine when recombination events have occurred during the evolution of T. cruzi. We analysed differences within and between T. cruzi subgroups in DNA sequences that evolve at different speeds. We found multiple recombination events, several of which were very recent in evolutionary terms. We show that TcV and TcVI most likely originated as a result of human activities that promoted mixing between subgroups, suggesting a continuing risk for emergence and spread of new genotypes generated by sexual reproduction. We also found evidence that sexual reproduction in T. cruzi could involve mechanisms different from those seen in multi-cellular eukaryotes.
PMCID: PMC3191134  PMID: 22022633

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