Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5041-5045, October 1979 Biochemistry A new endogenous primate type C virus isolated from the Old World monkey Colobus polykomos (retrovirus/genetic transmission/molecular hybridization) STEPHEN A. SHERWIN AND GEORGE J. TODARO Laboratory of Viral Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, NMaryland Communicated by Robert J. Huebner, July 20, 1979 ABSTRACT A new, genetically transmitted retrovirus has virus from the Old World monkey Colobus polykomos. This been isolated from the Old World monkey Colobus polykomos. virus is designated CPC-I for C. polykomos, type C, first isolate. This virus, designated CPC-1, is readily transmitted to both fe- line and human cells in culture. Nucleic acid hybridization CPC-1 is partially related by both antigenic and nucleic acid studies reveal that there are 50-70 copies of the CPC-1 genome hybridization criteria to the previously isolated MAC-1 virus in colobus cellular DNA. Related virogene sequences can be of stumptail monkeys. The virus is the first endogenous type detected in the DNA of all other Old World monkeys, as well C virus isolated from the Colobinae and appears to be analogous as in the DNA of at least one ape species, the chimpanzee, in- to the type C viruses of macaques, though the two subfamilies dicating that this virus has been genetically transmitted in pri- have been genetically separated mates for 30-40 million years. CPC-1 is partially related to the for nearly 20 million years. type C virus previously isolated from stumptail monkeys (MAC-1). These two viruses have nucleic acid sequence ho- MATERIALS AND METHODS mology, antigenic crossreactivity in their major viral structural protein, and a very similar host range in vitro. CPC-1 and MAC-1 Cell Culture and Virus Isolation. The cell lines used in these therefore belong to the same class of genetically transmitted experiments include a human carcinoma cell line A549 (14) that primate type C viruses and, as such, represent the first example has been unusually permissive for replication of various type in primates of analogous endogenous retroviruses isolated from C viruses; rhesus lung DBS-FRhL-1 (15); African green monkey two distantly related species. VERO, bat lung Tb-1-Lu, and mink lung MV-1-Lu from the Genetically transmitted retroviruses have previously been American Type Culture Collection; canine thymus Cf2Th from isolated from both New World and Old World monkeys (1-9). the Naval Biomedical Research Laboratory (Oakland, CA); These isolates have been classified as type C or type D viruses NIH/3T3 (16); and domestic cat embryo FEF from Peter based on morphologic properties and can be subdivided into Fischinger (National Cancer Institute). A primary culture of several distinct classes according to both antigenic and nucleic normal kidnev fibroblast cells obtained from a specimen of C. acid hybridization criteria (8, 10). Endogenous type C viruses polykomos was seeded with a variety of these indicator cell have thus far been isolated from the New World species owl lines. After 2 days, the cell mixtures were exposed to 5- monkey (Aotus trivirgatus) (7) and from Old World species, bromodeoxyuridine (100 ,ug/ml) for 24 hr. The cells were then including stumptail monkey (Macaca arctoides) (8), rhesus maintained in Dulbecco's modification of Eagle's medium monkey (Macaca mulatta) (9), and baboon (Papio anubis) supplemented with 10% fetal calf serum and were transferred (1-3); endogenous type D viruses have been isolated from the every 2 weeks by use of 0.1% trypsin in phosphate-buffered New World species squirrel monkey (Saimiri sciureus) (4, 5) saline. Cultures were screened at 3- to 4-week intervals over an and the Old World species langur (Presbytis obscurus) (6). To 8-month period for the presence of type C virus by assaying date, there have been no convincing isolations of endogenous supernatants for reverse transcriptase activity. type C or type D viruses from higher apes or humans. Viral Polymerase Assays. Culture supernatants were assayed The endogenous type C viruses isolated from Old World for reverse transcriptase activity with a polyriboadenylate monkeys include two distinct groups: the numerous closely template, an oligodeoxythymidilatel2-18 primer, and 0.6 mM related isolates from baboons (1-3) and the two highly similar manganese chloride as described (17). Antiviral polymerase isolates from macaques (8, 9). These two groups of viruses antibody inhibition studies were performed by published represent completely unrelated classes of genetically trans- methods (18). mitted retroviruses and are both represented in multiple copies Viral Structural Protein Assays. Competitive radioimmu- in the genomes of all Old World monkeys (8-12). Whereas the noassays for the presence of different structural proteins in viral baboon virus group is readily isolated from various species and extracts were performed as described (18). An assay for the p26 tissues of normal baboons (13), the two macaque viruses protein of MAC-1 virus (10) has been developed with a high- (MAC-1 and MMC-1) were both isolated in single, long-term titered antiserum from a goat inoculated with whole, disrupted experiments after multiple attempts had failed to yield virus MAC-1 virus. (8, 9). Molecular Hybridization. DNA and RNA were extracted The Old World monkeys can be divided into two subfamilies, from tissues and cell lines as described (11). 3H-Labeled DNA the Cercopithecinae, which includes baboons, macaques, and transcripts of CPC-1 virus that had been disrupted with Triton several other species, and the Colobinae, which includes the X-100 were prepared in an endogenous reverse transcriptase colobus and langur. In this paper, we report the isolation in a reaction and partially purified by sedimentation in alkaline 7-month cocultivation experiment of an endogenous type C sucrose (19, 20). Transcripts ranging in size from 12 S to 16 S in alkaline sucrose (2500-5000 nucleotides) were hybridized The publication costs of this article were defrayed in part by page to 2-4 mg of DNA or RNA per ml in the presence of 10 mM charge payment. This article must therefore be hereby marked "ad- Tris-HCl, pH 7.4/0.75 M NaCl/2 mM EDTA/0.05% sodium vertisement" in accordance with 18 U. S. C. §1734 solely to indicate dodecyl sulfate. Hybridizations were initiated by heating at this fact. 100°C for 10 min; the mixtures were then incubated at 650C 5041 Downloaded by guest on October 4, 2021 5042 Biochemistry: Sherwin and Todaro Proc. Natl. Acad. Sci. USA 76 (1979) for various lengths of time. Hybrids were detected with the Table 1. Hybridization of CPC-1 [3HJDNA transcripts to various single-strand-specific S1 nuclease (21). Less stringent hybrid- primate cellular DNAs ization conditions (12, 22) using a higher salt concentration (1.5 % hybridization* M NaCl) and a lower hybridization temperature (60'C) were 650C, 0.75 M 600C, 1.5 M performed where indicated. Cot values were calculated ac- Species NaCl NaCl cording to the method of Britten and Kohne (23) and corrected to a monovalent cation concentration of 0.18 M (25). [Cot is the Old World monkeys initial concentration of nucleic acid (mol/liter) multiplied by Colobinae time (sec).] Colobus: C. polykomos 96 92 RESULTS C. guereza 100 97 Langur 10 16 Isolation of CPC-1 Virus. A primary culture of normal Cercopithecinae kidney cells was obtained from a specimen of C. polykomos. Stumptail monkey 18 33 After 3 weeks in culture, these cells were seeded with ap- Pigtail macaque 19 34 proximately 106 cells of a variety of indicator lines. Two days Baboon 21 34 later the cell mixtures were exposed to 5-bromodeoxyuridine Patas 20 30 for 24 hr at a concentration of 100 ,gg/ml. The cell mixtures Mangabey 17 33 were subsequently tested at monthly intervals for the presence Vervet 15 29 of type C virus by assaying culture supernatants for reverse Apes transcriptase activity. After approximately 7 months, the culture Gibbon <1 5 containing human carcinoma A549 cells became positive for Orangutan <1 5 reverse transcriptase activity. This viral activity was subse- Chimpanzee 10 22 quently designated CPC-1 for C. polykomos type C virus. No Humans 1 7 such enzyme activity was detected in cultures containing bat New World monkeys Tb-i-Lu cells or canine Cf2Th cells (data not shown). Squirrel monkey <1 7 The CPC-1 viral polymerase activity required manganese Howler monkey <1 6 as its divalent cation; no significant enzyme activity was de- Owl monkey 2 NT tected when magnesium was substituted for manganese in the Prosimian Galago <1 8 reaction mixture. CPC-1 polymerase activity was fully inhibited Nonprimates by a broadly reacting antiserum to the polymerase of cat Cat 3 8 RD-114 virus, a serum previously shown to inhibit the poly- Sheep <1 6 merase of most type C viral isolates (data not shown). In these Squirrel <1 NT respects, therefore, CPC-1 reverse transcriptase activity appears Guinea pig <1 5 virus. to resemble that of a mammalian type C * Hybridization was determined by S1 nuclease digestion with CPC-1 Is an Endogenous Virus of the Colobus Monkey. [3H]DNA transcripts of CPC-1 virus that were 12 S-16 S in size. In order to determine whether CPC-1 is an endogenous retro- Reactions were carried out to a Cot of >103 at the indicated tem- virus of colobus monkeys, a DNA transcript of the virus grown peratures and salt concentrations. NT, not tested. in the human carcinoma cells was prepared in an endogenous reverse transcriptase reaction and hybridized to DNA extracted in other Old World monkey species that are close relatives of from the tissues of colobus monkeys and other primates.