Viruses, genes, and cancer

Viruses, genes, and cancer Viruses, genes, and cancer Viruses, genes, and cancer Viruses, genes, and cancer

Bacteriophage and Prokaryote Transduction

Bacteriophage lysis wasfirst observed in 1915 by the British microbiologist (Twort

Although Twort reported a filterable antibacterial factor, his findings were inconclusive, and Jules Bordet maintained that the lytic effect arose from an enzyme rather than a transmissible agent In investigations of dysentery among Allied troops, Félix d’Hérelle at the Institut Pasteur observed lysis of Shigella dysenteriae by a replicating filterable agent, and whether he knew of Twort’s report remains unclear; he did not cite it D’Hérelle was the first to establish multi-passage phage cultures, to quantify the extraordinarily high infection titers, and to coin the terms bacteriophage and plaque He believed phage could be used to cure sepsis, though his optimism was tempered by the difficulties of treatment, including the emergence of bacterial resistance, which helped phage therapy fall out of fashion in the West, while it continued to be promoted in the Soviet Union Today, phage therapy is experiencing a renaissance (Cisek et al 2017).

Nearly a century after D’Hérelle’s discovery, bacteriophages helped inaugurate the field of molecular genetics in the post–World War II era, led by Max Delbrück and the Phage Group Although lysogeny was not the group’s main focus, later work at the Institute Pasteur under André Lwoff advanced phage biology, revealing that lysogenic phages could mediate transduction of host genes These shifts—from phage genetics to gene transfer mechanisms—formed the core foundations of modern molecular genetics and phage biology.

Since 1953, DNA has been recognized not only as the central molecule of heredity but also as a practical tool in molecular genetics and gene regulation (Ptashne, 2004) Alongside plasmid transfer, DNA emerged as a major mechanism for horizontal gene transfer, enabling the exchange of host sequences among prokaryotes (Lane, 2015).

Forty years after Woese and Fox discovered the Archaea in 1977, the monophyletic origin of life forms is recognized for the evolutionary roots of prokaryotes, yet the extensive horizontal gene transfer among Archaea and Bacteria confounds attempts to draw accurate linear phylogenies Phage research remains highly relevant to modern molecular biology, providing models for evolutionary theory and enabling real-time experiments The discovery of DNA restriction enzymes emerged from phage host-range restriction, and the CRISPR-Cas9 gene editing mechanism has its origins in the bacteria’s natural defense against invading phages.

The Debt of Tumor Virus Research to Phage Genetics

The linear pedigree of tumor virus research unfolded more clearly than the prokaryote evolution described above At Caltech in the 1950s, Renato Dulbecco adapted the bacteriophage-based plaque techniques of the Phage Group to the growing practice of animal cell monolayers, yielding plaque assays for lytic viruses (Dulbecco 1952) and transformation assays for DNA tumor viruses (Vogt and Dulbecco 1960) His associate Harry Rubin studied Rous sarcoma virus (RSV) and, with his student Howard Temin, developed a quantitative transformation assay for RSV (Temin and Rubin 1958) After Rubin moved to Berkeley, Peter Vogt joined his laboratory and detected a non-transforming, replication-competent Rous-associated virus (RAV) in stocks of RSV (Rubin and Vogt 1962).

Temin (1960) observed RSV-transformed cells with an elongated, fusiform morphology that bred true according to the RSV variant used to infect them, and these cells were later shown to carry mutations in the C-terminus of the src gene This finding led Temin (1962) to distinguish cell transformation from viral replication, proposing that the virus carries genetic information that persists in host cells and influences their phenotype, an idea he likened to a lysogenic prophage In the same year, Svoboda observed that rats inoculated at birth with RSV non-productively still harbored the virus in adult tumors, supporting the persistence of a latent genome A year later, Crawford and Crawford (1961) demonstrated that the RSV genome is composed of RNA, not DNA Temin then postulated the provirus hypothesis, proposing that RSV persists as a latent genetic element within host cells.

Figure 1 shows scanning electron micrographs of cells transformed by the Rous sarcoma virus: panel a displays wild-type cells with a round morphology, while panel b shows mutant fusiform cells The infected cells form a DNA copy of the viral genome that integrates into the host genome, a concept central to the provirus hypothesis described by Temin in 1964 Although indirect evidence supported this hypothesis, it remained speculative until the discovery of reverse transcriptase by Temin and Mizutani (1970) and Baltimore (1970), which demonstrated how RNA viruses can generate DNA copies that anchor into the host genome.

Fifty-Five Years of Peter Vogt ’ s Contributions to Retroviruses and Oncogenes

Although Peyton Rous discovered his eponymous virus over 100 years ago (Rous

Although the transforming function could be titrated on the chick chorioallantoic membrane of chick eggs (Keogh 1938), it was not until Temin and Rubin established a quantitative in vitro virus assay in 1958 that RNA tumor virus research really blossomed (Weiss and Vogt 2011) Peter’s first paper on RSV showed that the Bryan high-titer strain used in the USA consisted of two components: a virus that transformed chick embryo fibroblasts and an accompanying avian leukosis virus (Rubin and Vogt 1962) This finding led to the discovery that this strain of RSV was replication-defective, missing the env gene encoding the envelope glycoproteins, which were provided by the helper leukosis virus (Hanafusa et al 1963).

Research from 1963 established the determinants of antibody neutralization specificity and RSV host range by exploring how different cell surface receptors influence infection (Ishizaki and Vogt, 1966; Vogt and Ishizaki, 1965) European RSV strains were non-defective and carried the src gene in addition to replication genes Strains such as B77, Carr-Zilber, Prague, and Schmidt-Ruppin were derived from RSV that had been used to induce non-productive tumors in rats and were rescued by inoculation into chickens or by co-cultivation with permissive chicken cells (Svoboda, 1966) The genome structures of these RSV strains were later depicted by Vogt and Hu (1977).

Replication-competent leukosis virus: LTR-gag-pol-env-LTR

Replication-defective RSV: LTR-gag-pol-src-LTR

Replication-competent RSV: LTR-gag-pol-env-src-LTR

These seminal studies clarified the distinction between replication genes and oncogenes, as demonstrated by Toyoshima and Vogt (1969), Duesberg and Vogt (1970), and Martin (1970) The work culminated in the demonstration of the host origin of oncogenes (Stehelin et al 1976) and led to the identification of the Src protein by Brugge and Erikson.

Identified in 1977 as the first example of a tyrosine kinase (Hunter and Sefton 1980), this finding revealed that many retroviral oncogenes have a cellular origin The broader pattern of retroviral oncogenes arising from cellular genes has since been reviewed by researchers including Steven Martin (2004), Peter Vogt (2012), Klaus Bister (2015), and Harold Varmus (2017) in his memoir on tumor viruses and cancer cell biology.

In the same year that Peter Vogt was appointed to CTMI’s Editorial Board, I published my inaugural virology paper detailing the release of infectious respiratory syncytial virus (RSV) particles with novel envelope properties, in the apparent absence of a helper virus.

Weiss (1967) To my surprise, and with gratification, a similar paper by Peter Vogt appeared a month later (1967) I was still a doctoral student of Michael Abercrombie, who had discovered contact inhibition but was not a virologist, and my mentor in Abercrombie’s laboratory, Warren Levinson, had returned to San Francisco I felt nervous because my findings seemed to contradict what was known about the defectiveness of RSV, and Peter’s paper helped me gain confidence.

I remained confident in my findings Moreover, Peter’s study was more elegant, showing that the virus he labeled RSV(0) preferentially infected Japanese quail cells rather than chick cells This work represented the first demonstration of xenotropism—the phenomenon later recognized in murine leukemia viruses (Levy1978) Our 1967 papers provided the initial steps toward the evidence that led to the elucidation of endogenous retroviruses, a history I have described elsewhere (Weiss2006).

2 Acquisition of Host Genes by Viruses

Oncogenesis by Simple Retroviruses and Transduction of Oncogenes

Leukemogenic retroviruses lacking oncogenes integrate at nearly random sites, with a preference for open chromatin regions in the host genome Among the millions of infected cells, some proviruses insert next to proto-oncogenes and activate their expression by leveraging promoter and enhancer elements within the retroviral long terminal repeat (LTR).

Initial studies defined c-myc in avian lymphoid leukosis (Hayward et al., 1981), highlighting the transduction of cellular oncogenes as an early mechanism (Vogt, 2012; Varmus, 2017) In avian lymphoid leukosis, activation of c-myc appears sufficient to drive B-cell leukemogenesis in the bursa of Fabricius, whereas gamma-retroviruses such as murine and feline leukemia viruses require more complex recombination events between the transmitted retrovirus and related endogenous genomes before the recombinant retrovirus activates cellular oncogenes (Rosenberg and Jolicoeur, 1997) For example, in cats, feline leukemia virus subtype A (FeLV-A) is the major transmissible agent, but most leukemia cases involve recombination with an endogenous retroviral genome encoding a B-env subtype, enabling leukemogenesis (Roy-Burman et al.).

1995) Multiple interactions occur between exogenous and endogenous LTRs and envregions giving rise to variants with different pathogenic attributes (Neil et al. 1991; Bolin and Levy2011; Stewart et al.2011).

Retroviral oncogenes show no shared structural features and act at diverse points in cellular signaling and regulation, including transcriptional regulators such as myc and jun, a concept studied by Peter Vogt Consequently, transduction of oncogenes by retroviruses does not depend on sequence homology; it occurs through read-through transcription and the packaging of RNA transcripts into virions, followed by reverse transcription and integration into the genome of the next infected cell via normal replication events (Vogt 2012) Apart from European strains of RSV passed through mammals, oncogene-bearing retroviruses appear replication-defective and have been rescued through aberrant splicing All that is required in the transcript is a contiguous virion packaging signal—the 5′ end of gag—with the host sequence.

There is little selective advantage for a virus to carry oncogenes beyond the observation that tumor cells tend to be more permissive to retroviral replication In fact, evidence that oncogene-bearing retroviruses are naturally transmitted from host to host is scant, unless they are brought to the attention of pathologists and virologists who deliberately propagate them An exception is the cyclin oncogene found in replication-competent epsilon-retroviruses in fish, but it is probably not of host origin (Rovnak and Quackenbush 2010) Oncogene transduction appears restricted to the simple retroviruses, namely alpha- and gamma-retroviruses, and it remains a puzzle why cellular gene incorporation into beta-, delta-, spuma-, and lentiviruses has not been observed to date.

Retroviral transduction of host oncogenes occurs across species, including primates, with oncogene-bearing viruses arising in outbred animals such as cats and chickens and inbred mouse strains In the waning days of my time in Peter Vogt’s lab, I spent two weeks at a chicken abattoir collecting solid tumors identified by veterinary inspectors along the processing line, finding about one tumor per 400 broiler chickens under six months old I left before I could determine what fraction of these tumor-bearing birds were infected with avian leukosis or whether the tumors carried transduced oncogenes Despite decades of work, there remains limited quantitative evidence of oncogene transduction in any species, though Miles and Robinson (1985) reported frequent transduction of the c-erbB oncogene in chicks with erythroblastosis after experimental inoculation with RAV-1 New oncogenes emerged in avian sarcomas, including jun, discovered by Vogt’s group as a pivotal transcriptional regulator Overall, the study of retroviral oncogenes has profoundly influenced cancer biology because many oncogenes later implicated in human cancer were first identified in animal retroviruses (Varmus 2017).

Why Is Retroviral Transduction not a Major Driver of Virus

of Virus or Host Evolution?

Retroviruses can both transduce host genes and invade germ lines across species, which raises the question of why they have not evolved into widespread, horizontally transferred transducing agents Notably, alpha- and gamma-retroviruses readily incorporate a variety of oncogenes and probably capture additional gene transcripts beyond what tumor-cell clonal expansion reveals If lysogenic bacteriophages are key drivers of prokaryotic evolution, researchers wonder why retroviruses do not fulfill a similar role in shaping vertebrate evolution.

Most oncogene-bearing retroviruses in birds and mammals are replication-defective and depend on helper viruses for successive replication cycles However, they can propagate in vivo and spread to new hosts if the concentration of helper virus is sufficient As with hepatitis delta virus and adeno-associated viruses, which rely on helpers, and plant partitiviruses with split genomes packaged in separate particles, defective oncogene-bearing retroviruses can still be successful pathogens Even if they do not spread contagiously through a host population, a single germ-line integration could achieve hereditary transduction Moreover, strong selective advantages for the host that favor transduction would likely drive the emergence of replication-competent transducing viruses, much as replication-competent strains of RSV have appeared.

Transduction limited to the same host species would exert little selective pressure for horizontal transmission, since it offers no clear advantage over sexual reproduction However, the widespread phenomenon of xenotropism and infection of unrelated germ-lines shows that retroviruses could be highly effective at enabling the lateral transfer of host sequences This broader potential for cross-lineage genetic exchange is discussed in Section 3.3.

A major limitation of retroviral transduction is that it relies on reverse transcription of spliced or partially spliced RNA, causing the inserted sequences in the new host to behave like pseudogenes mediated by retrotransposons If the intron-associated regulatory elements and micro-RNA sequences are missing, regulating the transduced gene’s expression can be difficult Nevertheless, introns can be gained during evolution, so this barrier is not insurmountable.

It may be that we have not probed deeply enough for evidence of horizontal gene transfer by retroviruses When such transfer occurs between distantly related hosts, whole-genome sequencing should reveal genes or pseudogenes that are more similar to the donor species—from which the retrovirus arose—than to the existing homologs in the recipient host Detecting this signal would indicate interspecies viral gene exchange and illuminate how retroviruses shape genome evolution across species boundaries.

Incorporation of Host Genetic Sequences into Complex Viruses

Retroviral oncogenes were first shown to be derived from host genes (Stehelin et al 1976), but the gene content of large, complex viral genomes remained poorly understood With the advent of whole-genome sequencing and bioinformatics, it has become possible to interrogate large DNA virus genomes for sequences hijacked from ancient and recent hosts and repurposed (exapted) to novel viral functions, shedding light on how viruses evolve by co-opting host genetic material.

fine tune the virus’s life style, whether it be replication, latency or immune evasion.

Krupovic and Koonin (2017) argue that key viral components, such as capsid proteins, originated from host cells This discussion, however, shifts focus to host-derived acquisitions that were later imported into viral genomes after viruses had already become established as replicating entities These late integrations reveal how viruses can co-opt host material long after their initial emergence, expanding their genomic repertoire through horizontal transfer from the host to the virus.

Herpesviruses, poxviruses, and insect polydnaviruses carry numerous genes derived from their hosts, reflecting long virus–host coevolution Viruses with substantial packaging capacity have captured host genes and modified them to serve viral functions Although poxviruses replicate in the cytoplasm, this has not prevented them from adopting multiple host genes Importin 7 mediates DNA trafficking between the nucleus and cytoplasm, potentially facilitating genetic exchange between virus and host Some viral genes contain introns, indicating horizontal transfer of host DNA rather than transcripts derived from reverse transcription of host RNA.

The extent of host gene invasion by large DNA viruses can be understood by comparing viruses within the same group that diverged millions of years ago; for example, Epstein-Barr virus and Kaposi’s sarcoma–associated herpesvirus share homologies among their core replication genes but show extensive differences in host-derived genes acquired over time Comparative genome analysis across human herpesviruses reveals multiple host contributions during virus evolution (Holzerlandt et al 2002), as depicted in Fig 2 Immunomodulatory genes are essential to the viruses’ in vivo life cycle, yet they tend to be lost during extensive in vitro passage, as seen in standard laboratory strains of cytomegalovirus and in the evolution of modified Vaccinia Ankara from wild-type Vaccinia virus Several acquired genes of oncogenic herpesviruses may contribute to oncogenesis, including chemokine homologs such as v-mip studied in Kaposi’s sarcoma–associated herpesvirus.

MicroRNA sequences are present in some viruses, with their precursors likely derived from cellular miRNAs Viruses can also manipulate host miRNA pathways to benefit their own replication and survival, such as Epstein-Barr virus proteins EBNA3A and EBNA3C that modulate c-myc regulation in B cells by inducing the expression of miR-221 and miR-222 in transformed B cells The remarkable evolution of polydnaviruses in parasitic wasps exemplifies a bidirectional genetic exchange between virus and host These large DNA viruses can become endogenous elements within their hosts, reflecting deep viral integration into host genomes.

Amoebae participate in extraordinary gene exchange with their intracellular viral and bacterial parasites, including giant Mimiviruses that carry many host-origin genes and are themselves prey to virophages, smaller viruses that parasitize these giant viruses This suggests something special about amoebae as hubs of endosymbiosis and horizontal gene transfer Legionella species, intracellular parasites of amoebae and occasionally human alveolar macrophages, also acquire hundreds of host genes, illustrating parallel patterns of gene sharing in intracellular lifestyles Conversely, some hosts can absorb entire or partial viral and bacterial genomes, as shown by the transfer of endosymbiont Wolbachia into insect and nematode nuclear genomes, highlighting the lasting impact of endosymbiosis on genome evolution.

3 Acquisition of Viral Genes by Hosts

Viral Genomes in Host DNA: Retroviruses, DNA Viruses and RNA Viruses

Whole-genome sequencing has revealed that every vertebrate species examined harbors multiple endogenous retroviral genomes (ERVs) This widespread pattern is supported by Stoye (2012), Hayward et al (2015), and Magiorkinis et al (2015) Moreover, analyses shown in Figure 3 indicate that roughly 8% of human DNA originates from germ-line infections by retroviruses, underscoring the long-lasting impact of retroviral integration on the human genome.

Figure 2 shows genes in human herpesviruses that have human homologs, noting that some genes are common to all herpesvirus types while others have been acquired independently Genes are color-coded by functional class: green for DNA replication; dark blue for nucleotide repair and metabolism; light blue for enzymes; purple for gene expression regulation; yellow for glycoproteins; red for host–virus interactions; and black for unknown functions Within each gene box, diagonal lines indicate two copies per genome, vertical lines indicate three copies, and horizontal lines indicate up to ten copies This depiction is adapted with permission from Holzerlandt et al 2002.

(Griffiths 2001) and a larger proportion of our genome is represented by retro- transposons such as LINE elements (Cordaux and Batzer 2009; Rebollo et al.

Endogenous retroviruses (ERVs) are better seen not as relics of an ancient RNA world converted into genes, but as fossils of highly evolved, sophisticated viruses that bear clear hallmarks of ancient infection The detection of endogenous spumavirus genomes in multiple hosts and the discovery of an endogenous single-copy delta-retrovirus genome in bats show that all seven retrovirus genera have endogenous counterparts The defective delta-retrovirus is distantly related to human T-cell leukemia virus and bovine leukosis virus and probably diverged more than 25 million years ago, while foamy viruses entered the germ-line even earlier.

Some retroviruses display promiscuous integration when DNA is available, and avian reticuloendotheliosis virus (REV) not only inserts into the host chromosomal genome but also into co-infecting DNA viruses in the same birds—Marek’s disease herpesvirus and fowlpox virus REV is a gamma-retrovirus of mammalian origin with related endogenous viruses in mongooses, and it has invaded birds and avian DNA viruses in the recent evolutionary past Niewiadomska and Gifford (2013) argue that REV was introduced iatrogenically from mammals to chickens and turkeys through administration of live attenuated viral vaccines Although 25 years have passed since the initial report of REV integration in a DNA viral genome (Isfort et al 1992), there is no known integration of other retroviruses into DNA viral genomes, suggesting this may be a special property of REV.

Fig 3 Proportion of transposable elements in the human genome Lines and Sines are retrotransposons (adapted with permission from Cordaux and Batzer 2009)

Evolutionary Dynamics of Exogenous and Endogenous Retroviruses

2016) and debate continues whether these endogenous genomes are linked to disease.

Endogenous viral elements have been detected across vertebrates, including an endogenous gamma-herpesvirus in the prosimian tarsier (Aswad and Katzourakis 2014), and avian hepadnaviruses such as duck hepatitis virus—rapidly evolving DNA viruses that use a reverse transcription step in their life cycle; a zebra finch endogenous hepadnavirus appears to have colonized the passerine germline about 20 million years ago (Gilbert and Feschotte 2010) Beyond viruses, bacterial genomes have been incorporated into the genomes of multicellular eukaryotes (Dunning Hotopp et al 2007) The presence of chromosomally integrated DNA complementary to RNA virus sequences was first reported for measles virus (Zhdanov 1975) and, after a long gap, recalled (Weiss and Kellam 1997) when Klenerman et al (1997) detected cDNA of hepatitis C virus, followed by discoveries of bornavirus and filovirus sequences (Belyi et al 2010; Horie et al 2010; Taylor et al 2010) The reverse transcription and integration are linked to LINE retroposon elements (Belyi et al 2010), and experimental studies of vesicular stomatitis virus infection in vitro show synthesis and integration of cDNA dependent on LINE-1 expression (Shimizu et al 2014).

Germline inserts of RNA virus genome fragments were first detected in bornaviruses and filoviruses related to Marburg and Ebola Genome-wide searches in animals and humans have revealed complementary DNA sequences derived from RNA viruses embedded in the germline, highlighting a broader spectrum of endogenous viral elements than previously appreciated Bornavirus-derived cDNA is widely dispersed across animal genomes, and while their functional role for the host is not yet known, these endogenous bornavirus sequences are expressed in human tissues and may inhibit exogenous infection, akin to the immunity to superinfection conferred by some endogenous retroviruses It remains unclear whether bornaviruses are currently infectious for humans, since clinical detections may reflect expression of endogenous bornavirus elements rather than active infection Overall, the accumulation of viral sequences in host germline DNA constitutes a broader phenomenon than the endogenous retroviruses we have long recognized.

3.2 Evolutionary Dynamics of Exogenous and Endogenous Retroviruses

Parasite-host interactions are commonly framed as an evolutionary arms race, a notion formalized by van Valen in 1973 as the Red Queen hypothesis This idea is echoed in the quote from Through the Looking Glass: "It takes all the running you can do to keep in the same place," the Red Queen tells Alice, "If you want to get somewhere else, you must run at least twice as fast as that." Rapidly replicating pathogens can outpace their hosts by continuously escaping immune defenses and developing drug resistance Consequently, host-pathogen coevolution drives ongoing adaptations in immune strategies and treatments to counter evolving pathogens.

There is, however, a stark difference between the replication rate of exogenous viruses and that of their endogenous counterparts, as noted by Lee et al (2013) for retroviruses and by Gilbert and Feschotte (2010) for avian hepadnavirus. Exogenous retroviruses undergo millions of replication cycles, including the reverse transcription step which is not subject to repair of errors, whereas endogenous retroviruses are by definition part of the host and replicate at the pace of the host germline, at less than 1000th the rate of virus replication (Aiewsakun and Katzourakis2015) Retroviruses parked in the genome of one host may act as a hidden reservoir for infection to another.

Potentially pathogenic ERV and retrotransposons are controlled by host intra- cellular restriction factors (Sanz-Ramos and Stoye 2013; Goodier2016) In addi- tion, the Env glycoprotein of ERV can itself act as a restriction factor against exogenous infection by blocking receptors (Malfavon-Borja and Feschotte2015), a phenomenon that wefirst reported for avian ERV (Payne et al 1971) However, such restrictions often do not apply if the virus gains access to an unrelated host species and it is clear that replication-competent ERVs can make large leaps to infect distant host species For instance, the baboon ERV that horizontally crossed hosts to become an ERV (RD114) in cats (Benveniste and Todaro1974) retains its ability to replicate and to re-infect cats in vivo (Shimode et al 2015) This phe- nomenon of xenotropism led us to examine the potential infection hazard of porcine ERV in pig-to-human xenotransplantation (Patience et al.1997; Le Tissier et al.

Cross-species infection by replication-competent endogenous retroviruses is exemplified by gamma-retroviruses related to gibbon ape leukemia virus (GALV) GALV isolates were obtained from captive gibbons in Thailand, although this exogenous retrovirus has not been detected in wild gibbons ERV sequences related to GALV have been found in the DNA of several Southeast Asian rodents, including lineages in Australia and Indonesia The koala retrovirus (KoRV) is also closely related to GALV and was first observed in 1988, though analyses of preserved taxidermy specimens indicate KoRV has been present in koalas for at least 120 years, possibly longer KoRV is associated with leukemia, with the current epidemic appearing to be linked to envelope variants KoRV-B or KoRV-J coexisting with the original KoRV-A KoRV-A uses the Pit-1 cell surface receptor, like GALV and FeLV-B, whereas KoRV-J uses a thiamine transport receptor similar to FeLV-A.

It thus appears that an ERV of rodents has crossed host species at least twice, to become an exogenous pathogenic retrovirus in both gibbons and koalas Moreover,

KoRV is in the process of becoming endogenized in the koala germ line, as supported by Tarlinton et al (2006) and Ishida et al (2015) Given that many Muridae rodents carry related endogenous retroviruses (ERVs) (Alfano et al., 2016), it is likely that the GALV precursor originated in rodents and subsequently spread horizontally to koalas and apes, as illustrated in Figure 4.

Pathogenic and Bene fi cial Attributes of Endogenous Retroviruses

ERVs and other retrotransposons are subject to natural selection in the host, forming a two-edged scenario: the integration and expression of certain ERVs can be linked to disease, while the presence of transposable elements may also confer overall benefits to the host population (Rebollo et al 2012; Babaian and Mager 2016) Much debate surrounds the deleterious versus beneficial effects of ERVs (Moyes et al 2007; Stoye 2012), and ongoing research on new ERVs continues to illuminate their complex roles in genome evolution and host biology.

Endogenous retroviruses can act as reservoirs within a host for millions of years and occasionally emerge to invade unrelated species Gamma-retroviruses that are inherited as Mendelian genomes in Southeast Asian rodents have transferred horizontally to gibbons (GALV) and to koalas (KoRV) KoRV is becoming endogenous in its koala host Insertion events, as reported by Benveniste et al (1977), Kawakami et al (1978), and Tarlington et al (2006), may disrupt essential host genes (Chuong et al 2017) More recently integrated ERVs can be oncogenic, contributing to leukemia in mice and cats and to mammary cancer in mice (Rosenberg and Jolicoeur 1997; Ross 2010).

Whether human endogenous retroviral sequences (HERVs) play a causal role in cancer remains unresolved (Magiorkinis et al., 2015; Babaian and Mager, 2016; Kassiotis and Stoye, 2017) Notably, beta-retrovirus HERV-K (HML2) genomes have been implicated in testicular cancer, melanoma, and breast cancer Although certain HERV-K loci show higher expression in tumor tissue, this alone does not establish causality; tumor cells may provide a permissive environment that makes HERV-K expression a consequence rather than a driver of malignancy.

Human endogenous retroviruses can benefit the host by providing non-coding regulatory sequences and by encoding useful proteins For example, HERV-K is expressed in pre-implantation embryos and is linked with pluripotency, but becomes transcriptionally silenced during differentiation HERV-H elements and LINE sequences also play a key role in transcriptional regulation within pluripotent cells and influence early development ERVs can modulate host gene expression, as seen with the HERV-E insertion that drives salivary amylase expression in the parotid gland—an activation that arose in the primate lineage and expanded in hominids The HERV-E promoter and tissue-specific enhancers enable amylase expression outside the pancreas, a change that may have helped our ancestors adapt from a fruit-based diet to one that includes starch.

Role of Endogenous Retroviral Envelopes in the Placenta

Endogenous retroviruses (ERVs) offer a striking example of host benefit, with the Env protein driving cell fusion to form the syncytiotrophoblast in the mammalian placenta The idea that a HERV envelope could influence placental development has intrigued researchers since the early 1990s, and this concept has been notably advanced by the laboratories of Thierry Heidmann, John M McCoy, and Franỗois-Lọc Cosset.

In the human placenta, there is high expression of a defective human endogenous retrovirus with an open reading frame for env, known as ERV-3 (Boyd et al 1993) ERV-3 expression is tightly linked to the syncytiotrophoblast, supporting the idea that a functional retroviral Env glycoprotein could drive cell-to-cell fusion (Venables et al 1995) Lin et al (1999) demonstrated that transfection of ERV-3 into BeWo choriocarcinoma cells—a malignant variant of cytotrophoblast—induced cell fusion and differentiation into syncytiotrophoblast However, it was found that some humans lack the ERV-3 genome (de Parseval and …).

Research by Heidmann (1998) suggests that gestation relies on a healthy placenta Gorillas lack ERV-3, as reported by Hervé et al (2004) Nevertheless, the central idea that endogenous retroviruses (ERVs) drive placental differentiation remains viable, though it may involve different ERV families across species Blond et al contribute to this view by highlighting the evolutionary diversification of ERV-mediated placental mechanisms.

Studies by Mi et al (2000) showed that the HERV-W envelope (Env) protein can induce syncytiotrophoblast fusion, and they coined the term syncytin for this envelope glycoprotein Building on this, Lavillette et al (2002) demonstrated that HERV-W Env interacts with several amino-acid transporters that are already known to act as receptors for beta-retroviruses, thereby triggering membrane fusion Together, these findings establish syncytin as the functional HERV-W envelope glycoprotein essential for placental cell fusion.

The syncytin story grew more complex with the discovery of a second human Env glycoprotein, syncytin-2, encoded by HERV-FRD (Blaise et al., 2003) Low expression of syncytin-1 and syncytin-2 is linked to poor placental development and pre-eclampsia (Vargas et al., 2011) Moreover, different orders of placental mammals use Env glycoproteins from distinct endogenous retroviruses (ERVs) to drive trophoblast cell fusion, extending even to a proto-placenta in the marsupial opossum (Lavialle et al., 2013; Cornelis et al., 2015).

If placenta evolution were a single, monophyletic event, the repeated recruitment of different endogenous retroviruses (ERVs) to drive trophoblast fusion into a syncytium remains puzzling Imakawa et al (2015) proposed a baton-pass hypothesis in which successive ERV variants assume the cell-fusion function, producing diverse placental architectures and boosting reproductive success in placental mammals They argue that ERVs have replaced older mediators of trophoblast fusion rather than reflecting a single ancestral mechanism This view is supported by the observation that several ERV genes encoding syncytins became endogenous in their host genomes only within the last 12–80 million years, more recently than the initial evolution of the mammalian placenta In humans, redundancy among retroviral drivers of trophoblast fusion helps explain why ERV-3, HERV-W, and HERV-FRD can each mediate cytotrophoblast fusion to form the syncytiotrophoblast.

HERV Env may have a dual role in the placenta: it can act locally to suppress immune responses at the maternal-fetal interface (the syncytiotrophoblast) to protect the fetus from maternal rejection, and it may also induce trophoblast fusion (Denner 2016) The immunosuppressive domain in the transmembrane envelope protein is present in ERV-3 and HERV-Wenv (Boyd et al 1993; Lavialle et al.).

Syncytins, including HERV-W Env, retain their virus entry capacity and can functionally pseudotype retroviral vectors, highlighting their ongoing role in envelope-mediated entry HERV-W is expressed in multinucleated macrophages such as osteoclasts, but there is no evidence that other syncytial tissues, such as striated muscle, use ERV-derived syncytins for cell fusion Nevertheless, placental mammals have repurposed ERVs by harnessing their envelope proteins to promote cell fusion, exploiting retrovirus envelope–cell surface receptor interactions to provide host-beneficial fusogenic functions.

4 Endosymbiont Organelles in Eukaryotic Cells and TheirHorizontal Transfer in Cancer

Evolution of Complex Cells and Their Organelles

Peter Vogt’s appointment to the CTMI Editorial Board coincides with the birth of the endosymbiotic theory—the idea that eukaryotic cells originated from the merging of prokaryotes to form organelles Proposed by Lynn Margulis (then publishing as Sagan) in 1967, the concept was initially met with considerable skepticism Margulis was known for courting controversy, including the view that HIV does not cause AIDS, arguing that AIDS is essentially syphilis masquerading under a new name in her work (Margulis et al 2009).

Endogenous envelope glycoproteins function in the human placenta as syncytins Indirect immunofluorescence reveals the ERV-3 envelope glycoprotein in the syncytiotrophoblast of a full-term placenta (Venables et al., 1995) In Cos cells, a reverse-oriented HERV-W syncytin-1 construct fails to promote fusion, while a correctly oriented HERV-W syncytin-1 drives syncytium formation (Mi et al., 2000).

Current views hold that mitochondria, chloroplasts, cilia, and the nuclear membrane have distinct origins but coalesced to form eukaryotic cells through long-standing endosymbiosis, a process whose exact steps remain debated During this endosymbiotic era, many genes relocated to the nuclear genome as mitochondrial DNA was reduced If subcellular organelles in eukaryotes are monophyletic, to what extent does lateral gene transfer occur between species? In animals, mitochondrial genomes have co-evolved with the nuclear genome, yielding concordant phylogenies, whereas higher plants show widespread lateral mobility of mitochondria and plastids This divergence may reflect mitochondrial variation driving the evolution of sex and the germline-soma distinction in metazoans, which is not present in higher plants.

Mitochondrial transfer between animal cells is illustrated by research on transmissible tumor cells, an area I joined a few years ago My interest was sparked by a 1983 note from Hayes and colleagues, published just before the discovery of HIV, which speculated that canine transmissible venereal tumor (CTVT) might serve as a model for Kaposi’s sarcoma in AIDS—a view later reconsidered after the HIV causative virus was identified (Chang et al., 1994).

Horizontal Spread of Cancer Cells

Some transmissible cancers spread not by oncogenic viruses but through the migration of the tumor cells themselves In Canine Transmissible Venereal Tumor (CTVT), marker chromosomes indicated that the transmissible agent is the cancer cell lineage rather than a virus A LINE-1 retrotransposon insertion near the c-myc gene was found to be unique to the tumor Forensic DNA markers confirmed the cellular transmission of CTVT and demonstrated that the tumor represents a single clone that has colonized dogs worldwide.

CTVT was first described in 1876 and played a pivotal role in early cancer research as the only tumor that could be experimentally transplanted between animals before the development of inbred lines; it likely emerged in an ancient dog breed about 11,000 years ago, making it roughly 2,000 times older than HeLa cells, and it has since accumulated thousands of somatic mutations, deletions, amplifications, and chromosome rearrangements, providing a unique model to study how a tumor cell clone can retain proliferative capacity despite extensive genomic changes Despite its parasitic nature, CTVT has not undergone massive genome reduction, since deletions of nonessential genes would have to occur on a case-by-case basis with housekeeping and specialty genes often interspersed The devil facial tumor disease (DFDT/DFTD) is also transmitted horizontally as a tumor cell in the Tasmanian devil, an endangered marsupial with relatively low genetic diversity, where two independent DFDT clones circulate, both of recent provenance Research has shown that some clam species harbor clonal tumor cells, including instances where the host species differs from the tumor’s origin, illustrating diverse transmissible cancers across taxa Transmission modes vary: in dogs the tumor spreads mainly through sexual contact, in devils via biting, and in clams through water filtered containing tumor cells The emergence of transmissible tumors remains rare and immune evasion is not fully understood, with a lack or downregulation of major histocompatibility antigens implicated in promoting tumor emergence; there are also human examples of horizontal transmission from donors to immunosuppressed transplant recipients, leukemia transmission in utero between fetuses sharing a placenta, and a naturally transmissible leukemia in Syrian hamsters spread by mosquitoes.

Colonization of Cancer Cells by Host Mitochondria

In investigating the most recent common ancestor of the tumor cell clone, researchers found conflicting signals between nuclear microsatellite DNA and mitochondrial DNA, with mtDNA implying a longer divergence; this led to the recognition of two major mtDNA clades in CTVT with distinct origins, a hypothesis later confirmed by Rebbeck et al 2011 It is now clear that host mtDNA has been acquired at least five times as the tumor clone passed through numerous canine hosts (Strakova et al 2016) Mitochondrial function may drive tumor progression, and recombination between mtDNA genomes has been observed in some CTVT tumors, linking mtDNA dynamics to pathogenic fitness Mutations in mtDNA can accrue during serial passage, and acquisition of host mtDNA may enhance the tumor’s persistence, suggesting that a globally spreading tumor as a somatic cell parasite has itself been colonized by host mitochondria via lateral transfer.

Future research should determine whether host mitochondria similarly colonize tumors in other transmissible cancer cells that have recently come to light, extending observations beyond the initial cases In particular, the tumor that colonized a different clam species from its origin (Metzger et al., 2016) provides a suitable system to search for evidence of cross-species transfer of mitochondria and other organelles, offering insights into mitochondrial dynamics and evolution in transmissible tumors.

Mitochondria have transferred many of their genes to the nuclear genome over evolutionary time, a process that may be mediated by Importin 7 Consequently, cytoplasmic organelles containing DNA are likely constrained to co-evolve with the nuclear genome, due to the interplay of non-coding RNA elements (Lane 2010; Danoya et al 2013; Vendramin et al.).

Although lateral transfer of mitochondria to distantly related species is unlikely, horizontal spread of mitochondria can occur within individuals and has been detected in cancer In an experimental murine system with tumor cells devoid of mitochondria, tumor progression was activated when mitochondria were transferred from host stroma (Tan et al 2015) Similarly, human tumor cells may acquire host mitochondria from endothelial cells (Pasquier et al 2013) As a consequence, tumor progression and relapse may involve colonization of tumor cells by external mitochondria.

‘fitter’mitochondria imported from the host.

Horizontal gene transfer is widespread across all life, with viruses embedding into host DNA and host genes moving into viral genomes Endogenous retroviruses can lie dormant in the germ line for millions of years and re-emerge as replication-competent viruses that can infect distantly related species Meanwhile, host cells may become transmissible malignant clones that outlive most vertebrate somatic cells, and these cells can be sequentially colonized by mitochondria from new hosts over multiple transplant generations These processes reveal far greater fluidity of genomes and cells in the eukaryotic world than was imagined fifty years ago.

Acknowledgments: I am deeply grateful to my early-career mentors—Warren Levinson, Jan Svoboda, and Peter K Vogt—for cultivating a stimulating intellectual environment and a rigorous approach to experimental research I also thank Klaus Bister, Ariberto Fassati, and Paul Kellam for their constructive discussions that enriched my work.

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