Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria Gloria Arriagadaa,b,1, Michael J. Metzgera,1, Annette F. Muttrayc, James Sherryc, Carol Reinischc, Craig Streetd, W. Ian Lipkind, and Stephen P. Goffa,2 aDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; bDepartamento de Ciencias Biologicas, Universidad Andres Bello, Viña del Mar 2520000, Chile; cEnvironment Canada, Water Science & Technology Directorate, Burlington, ON, Canada L7R 4A6; and dCenter for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY 10032 Contributed by Stephen P. Goff, May 30, 2014 (sent for review January 24, 2014) Bivalve mollusks of the North Atlantic, most prominently the soft involvement of an endogenous retrovirus or retrotransposon. shell clam Mya arenaria, are afflicted with an epidemic transmissi- Some studies have detected reverse transcriptase (RT) activity in ble disease of the circulatory system closely resembling leukemia. neoplastic clam tissues (14, 18–21), suggesting that a retroele- The disease is characterized by a dramatic expansion of blast-like ment or retrovirus might be involved in the disease process, but to cells in the hemolymph with high mitotic index. Examination of date searches for viruses and retroviral sequences from leukemic hemolymph of diseased clams revealed high levels of reverse tran- clams have not been successful (22). scriptase activity, the hallmark of retroviruses and retroelements. By deep sequencing of RNAs from hemolymph, we identified tran- Results scripts of a novel retroelement, here named Steamer. The DNA of Identification of a Novel Retroelement, Steamer. To test for the the element is marked by long terminal repeats and encodes a sin- presence of retroviruses or retroelement virus-like particles, we gle large protein with similarity to mammalian retroviral Gag-Pol assayed cell-free hemolymph from diseased and healthy clams proteins. Steamer mRNA levels were specifically elevated in dis- for RT activity, using a synthetic homopolymer substrate (23). eased hemocytes, and high expression was correlated with disease Hemolymph from diseased clams frequently exhibited high levels status. DNA copy number per genome was present at enormously of RT activity, 2- to 10-fold above the background activity ob- high levels in diseased hemocytes, indicative of extensive reverse served in hemolymph from healthy clams (Fig. 1A). To confirm transcription and retrotransposition. Steamer activation in M. are- that the RT activity was released by neoplastic hemocytes rather naria is an example of a catastrophic induction of genetic instability than other tissues, we cultured the hemocytes and determined that may initiate or advance the course of leukemia. the level of RT activity accumulated in the media (postcultured hemolymph). Hemocytes from diseased animals cultured in vitro retrotransposon | mobile genetic element | integration | released high levels of RT into the culture medium, 20- to 50- disseminated neoplasia | hemic neoplasia fold greater than culture medium of hemocytes from healthy animals (Fig. 1B). To identify the potential source of the RT he soft shell clam Mya arenaria is one of the most primitive activity, we cultured cells from a diseased clam with high RT Tspecies in the animal kingdom to manifest a leukemia-like activity, isolated total RNA from the culture medium, and used disease, variously termed hematopoietic, hemic, or disseminated 454 sequencing of cDNAs to generate a database of ∼200,000 neoplasia (reviewed in ref. 1). The disease is characterized by the sequence reads. Searching these sequences revealed 15 reads presence of abnormal, rounded, rapidly proliferating hemocytes with matches to retroviral protease, RT, or integrase sequences. GENETICS containing large pleiomorphic nuclei and multiple nucleoli. The – tumor cells are polyploid or aneuploid (2 4), exhibit abnormal levels Significance and cytoplasmic localization of the p53 tumor suppressor protein (5), and often express a 200-kDa cell surface antigen defined by monoclonal antibody 1e10 (6–9). The increase in DNA content can The soft shell clam in many areas of the North Atlantic is be detected by flow cytometry, and the severity of the disease can be afflicted with a fatal leukemia-like disease of unknown origin. established using histological methods. As the disease progresses, Leukemic cells from the diseased animals were found to release normal amitotic hemocytes are replaced by proliferating leukemia reverse transcriptase and to express high RNA levels of a pre- viously unknown member of the gypsy family of retroelements, cells that invade all tissues, with fatal consequences. A similar dis- Steamer. The DNA copy number of the element was increased ease has been described in several species of bivalves, including to enormously high levels in diseased cells, mediated by reverse oysters (Crassostrea virginica, Crassostrea gigas, Ostrea edulis), mus- transcription and integration into the host genome. The acti- sels (Mytilus edulis, Mytilus galloprovincialis, Mytilus trossulus, Mytilus vation of Steamer expression and transposition may initiate or chilensis), cockles (Cerastoderma edule), and clams (Macoma spp., accelerate the course of leukemia and constitutes a potential M. arenaria,andMya trunata) over a wide geographic distribution. diagnostic marker of the disease. Despite many reports describing some of its characteristics (1, 10), little is known about the onset and etiology of the disease. Author contributions: G.A., M.J.M., J.S., C.R., and S.P.G. designed research; G.A., M.J.M., Stressors such as pollution (1, 11), temperature (12), and over- A.F.M., J.S., C.R., and S.P.G. performed research; G.A., M.J.M., C.S., W.I.L., and S.P.G. crowding have been implicated in disease development. There is analyzed data; and G.A., M.J.M., and S.P.G. wrote the paper. evidence that the disease can be transmitted from infected to The authors declare no conflict of interest. uninfected individuals (13, 14), indicative of an infectious etiol- Data deposition: The sequence reported in this paper has been deposited in the GenBank ogy. Unfiltered hemolymph, lysed hemocytes (15), and even fil- database (accession no. KF319019). tered hemolymph isolated from BrdU-treated animals (16) were 1G.A. and M.J.M. contributed equally to this work. found to induce disease in healthy animals, raising the possibility 2To whom correspondence should be addressed. Email: [email protected]. of a filterable transmissible agent such as a virus. Induction of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. disease by the retroviral inducer BrdU (17) suggested the possible 1073/pnas.1409945111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1409945111 PNAS | September 30, 2014 | vol. 111 | no. 39 | 14175–14180 Downloaded by guest on September 26, 2021 Fig. 1. RT activity and isolation of Steamer DNA from hemolymph of leukemic M. arenaria.(A) RT activity in cell-free hemolymph of indicated leukemic or normal M. arenaria animals was determined by assay for incorporation of {32P}dTTP onto a homopolymer substrate (23). Spot intensity reports the yield of labeled DNA synthesized in vitro, quantitated by ImageJ and normalized to the average of normal values. (B) Hemocytes from the indicated animals di- agnosed as leukemic or normal (L or N) were cultured in vitro, and RT activity present in the postculture supernatant medium was determined as in A.(C) Alignment of selected sequences obtained by deep sequencing of cDNAs from a leukemic clam with a retroviral pol gene. PCR primers, forward (F) and reverse (R), are indicated. DNAs amplified by various primer pairs are indicated below the element diagram. (D) DNAs amplified in PCR reactions using cDNA obtained from leukemic clams as a template. Major amplified products are indicated by arrows at the right. (E) Schematic of Steamer genome annotated with characteristic retroelement features. The 5′ and 3′ LTR and the locations of the coding sequences for CA (capsid), NC (nucleocapsid), PR (protease), RT, RH (RNaseH), and IN (integrase) domains are indicated. Characteristic sequence features of each domain, and predicted primer binding site (PBS) and polypurine track (PPT) are indicated. RT-PCR reactions, using different combinations of primers based Expression of Steamer RNA Is Elevated in Diseased Hemocytes. To on these sequences and RNA preparations from cell-free he- test for expression of Steamer RNA transcripts, soft shell clams were molymph of a leukemic clam, yielded three long overlapping collected from Prince Edward Island (PEI) in Canada and di- DNA fragments (Fig. 1 C and D). The sequence of a complete agnosed according to hemocyte morphology. Total RNA was iso- copy of the retroelement containing these fragments (4,968 bp) lated from hemocytes of normal (n = 43) and moderately (n = 10) was obtained by genome walking using DNA from a healthy and heavily leukemic (n = 21) individuals, and the levels of Steamer animal (Fig. S1). This retroelement was named Steamer for the RNA were determined by quantitative RT-PCR (qRT-PCR) and common name of the host clam and also, by tradition in the normalized to a housekeeping RNA. Steamer RNA levels were transposon field, for a mode of transportation. generally low in the normal and moderately leukemic animals, al- The Steamer element contains a single long ORF with