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Home , Baboon, Cercopithecinae, Colobinae, Galago, Green monkey, Macaque

Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5041-5045, October 1979 Biochemistry A new endogenous type C isolated from the Old World 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 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 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 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 and appears to be analogous as in the DNA of at least one , the , in- to the type C of , though the two subfamilies dicating that this virus has been genetically transmitted in pri- have been genetically separated mates for 30-40 million . 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 of analogous endogenous retroviruses isolated from C viruses; rhesus lung DBS-FRhL-1 (15); African 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 (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 (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 or . 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 (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 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 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 , 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 - 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 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 <1 5 containing human carcinoma A549 cells became positive for <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 <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 <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 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. The the colobus, a further characteristic of a genetically transmitted results of these experiments are shown in Table 1. Hybridization virus. was detected by S1 nuclease digestion at two temperatures and The data in Table 1 also reveal that the CPC-1 transcript salt concentrations: "high stringency" conditions (65'C and 0.75 hybridized at low levels to the DNA of , but not M NaCl), at which only closely matched nucleotide sequences detectably to the DNA of other higher primates, including hybridize, and "low stringency" conditions (60°C and 1.5 M , , and humans. The hybridization to NaCl), at which more distantly related hybrids can be detected chimpanzee DNA was significantly higher than the nonprimate (22). As shown in Table 1, at either high- or low-stringency background levels at both high-stringency (10%) and low- conditions, the CPC-1 DNA transcript hybridized almost stringency (22%) conditions. This suggests that chimpanzees completely (>90%) to the DNA extracted from colobus monkey also contain CPC-1-related sequences in their genome despite tissues. There was no appreciable difference in the level of the evolutionary divergence of colobus monkeys and chim- hybridization to the DNA of the two different colobus species panzees, which occurred nearly 30-40 million years ago. In that were tested. In contrast, there were much lower levels of contrast, under the same hybridization conditions, no related hybridization at either high- or low-stringency conditions to virogene sequences could be detected in the DNA of the other the DNA of other primate species. These findings demonstrate apes tested and of various New World monkeys, , that CPC-1 was derived from endogenous virogene sequences and nonprimates. present in colobus monkey cell DNA. To further demonstrate that CPC-1 is an endogenous virus As indicated in Table 1, the CPC-1 transcript hybridized of colobus monkeys, we performed experiments to determine significantly to the DNA of other Old World monkey species. the number of copies of the CPC-1 genome in colobus DNA. At high-stringency conditions there was approximately 10% Endogenous retroviruses are typically found in multiple copies hybridization to langur DNA and between 15 and 21% hy- in the cellular DNA of the species of origin (25). Fig. 1 shows bridization to the DNA of Old World monkeys belonging to the the kinetics of hybridization of the CPC-1 transcript to colobus Cercopithecinae subfamily (baboon, patas, mangabey, vervet, cellular DNA and to the cellular DNA of another Old World and macaque). Moreover, at low-stringency conditions, there monkey, the stumptail macaque. As shown, the Cotl/2 for the was a significant increase in the level of hybridization (29-34%) hybridization to colobus DNA is approximately 30. Because the to the DNA of Cercopithecinae species. Thus, the CPC-1 C0t1/2 for the self-annealing of single-copy mammalian DNA transcript easily detects partially related virogene sequences under identical reaction conditions is 1500-2000 (see Fig. 1), Downloaded by guest on October 4, 2021 Biochemistry: Sherwin and Todaro Proc. Natl. Acad. Sci. USA 76 (1979) 5043

C

0 'M 60-

100 0.01 1.0 100 .C 40 Competing protein, Mg FIG. 2. Competition radioimmunoassays for viral structural proteins. (A) Interspecies assay for Old World monkey type C viral 20- p30 with goat antibody to MAC-1 and 1251-labeled M28 p30. (B) Species-specific assay for MAC-1 p26 with goat antibody to MAC-1 and 125I-labeled MAC-1 p26; (C) species-specific assay for M28 p30 with goat antibody to M28 and 125I-labeled M28 p30. The viral ex- 10" 10' lo2 103 104 tracts used as competing antigens were colobus CPC-1 (0), stumptail Cot, (moles X sec)/liter macaque MAC-1 (0), baboon M28 (-), and owl monkey OMC-1 (A). FIG. 1. Hybridization of the [3HJDNA transcript of CPC-1 virus to colobus and stumptail monkey DNAs. The percent hybridization was determined by S1 nuclease digestion and is normalized. The ac- These results demonstrate nucleic acid sequence homology tual percent hybridization for the homologous control was 94%. The DNAs tested were extracted from colobus (0), stumptail monkey (0), between CPC-1 and MAC-1. This is, in fact, confirmed by the humans (A), and sheep (A). The self-annealing of 3H-labeled non- reciprocal hybridization of the MAC-1 transcript to CPC-1 viral repetitive stumptail monkey cellular DNA is shown for reference RNA, which resulted in 29% hybridization (Table 2). (x). On the basis of the apparent nucleic acid homology between CPC-1 and MAC-1, it seemed probable that at least some of the one can estimate that there are approximately 50-70 copies of structural proteins of these two viruses would share a degree the CPC-1 genome in colobus cellular DNA. Moreover, the of antigenic relatedness. This hypothesis was tested in a series Cotl/2 for the hybridization of the CPC-1 transcript to stumptail of competition radioimmunoassays for known viral structural macaque DNA is also approximately 30, although the final proteins. Fig. 2 shows the results of some of these experiments. extent of hybridization is much lower (24%). This suggests that The CPC-1 viral extract was highly positive in the interspecies there are 50-70 copies of a partially related viral genome in the assay, which detects both M28 baboon viral p30 and MAC-I DNA of the stumptail macaque. This is the expected result, macaque viral p26 (Fig. 2A), thus confirming that CPC-1 can assuming that CPC-I is an endogenous colobus virus and that be detected in an assay for Old World monkey type C virus. partially related virogene sequences are present in the same CPC-1 extract was also positive in the species-specific assay for copy number in the DNA of other Old World monkeys. MAC-1 p26, an assay which does not score M28 p30 (Fig. 2B). CPC-1 Is Related to MAC-I, the Endogenous Type C Virus In this assay, the CPC-1 viral protein crossreacted in the com- of Stumptail Macaques. CPC-1 was tested for relatedness to petition assay but had a different slope from that of the ho- other previously isolated endogenous primate retroviruses by mologous MAC-1 control. This indicates that CPC-I contains hybridizing the CPC-1 transcript to the RNA of these viruses. a viral structural protein antigenically related to, but never- As shown in Table 2, the CPC-1 transcript hybridized 18% to theless distinct from, the p26 protein of MAC-1. In contrast, the RNA of MAC-1, the endogenous type C virus of stumptail neither CPC-1 nor MAC-1 viral extracts were detectable in the macaques, but did not hybridize to the RNA of other primate species-specific assay for baboon M28 p30 (Fig. 2C). type C viruses isolated from baboons and owl monkeys or of The relationship between CPC-1 and MAC-1 was further type D viruses isolated from langurs and squirrel monkeys. explored by comparing the in vitro host range of these two vi-

Table 2. Hybridization of CPC-1 [3H]DNA transcripts to RNA of Table 3. Comparison of host range of CPC-1 and MAC-1 endogenous primate retroviruses virus isolates reverse % hybridization with Supernatant [3H]DNA transcript transcriptase activity Species of of:t Indicator cell 6 wk after infection with:* origin Viral RNA* CPC-1 MAC-1 lines CPC-1 MAC-1 Colobus CPC-1, type C 100 29 Human A549 1028.1 230.4 Stumptail monkey MAC-1, type C 18 100 Bat Tbl-Lu 3.2 1.9 Baboon M28, type C 1 2 Rhesus DBS-FRhL-1 5.9 3.9 Langur PO-1-Lu, type D <1 2 Canine Cf2Th 5.2 4.4 Owl monkey OMC-1, type C <1 1 Cat FEF 51.1 212.3 Squirrel monkey M534, type D 2 3 Mink Mv-1-Lu 4.2 4.4 Green monkey VERO 2.7 2.0 * Viral RNA was obtained from heterologous cell lines producing Mouse NIH/3T3 2.4 5.3 these viruses: CPC-1, MAC-1, and M28 were grown on human A549 cells; PO-1-Lu and OMC-1 on bat Tbl-Lu cells; and M534 on * Indicator cell lines were infected with 1.0 ml of filtered supernatant DBS-FRhL-1 cells. of A549 cells producing either CPC-1 or MAC-1 viruses. Culture t The percent hybridization, as determined by S1 nuclease digestion, supernatants were subsequently assayed for reverse transcriptase is normalized. The actual percent hybridization for the homologous activity. Data are expressed as cpm X 10-3 of [3H]dTMP incorpo- control was >90%. rated. Values significantly above background are italicized. Downloaded by guest on October 4, 2021 5044 Biochemistry: Sherwin and Todaro Proc. Natl. Acad. Sci. USA 76 (1979) ruses. Table 3 shows the results of an experiment in which a variety of indicator cell lines were infected with filtered su- pernatants of A549 cultures producing CPC-1 and MAC-1. Both of these viruses can be easily transmitted within 6 weeks after PROSIMIANS infection to human A549 cells and to cat FEF cells. In contrast, the growth of both viruses appears to be restricted in a variety 60 Myr of other mammalian cell lines. These results suggest a possible NEW WORLD MONKEYS similarity in the viral envelope glycoproteins of CPC-1 and /40 MAC-I to the extent that this viral protein is an important de- OWL MONKEY TYPE C terminant of host range in vitro. Thus, CPC-1 and MAC-1 may SQUIRREL MONKEY TYPE D have a similar envelope glycoprotein as well as an antigenically related p26 structural protein. Both of these experimental OLD WORLD MONKEYS APES findings are in keeping with the observed nucleic acid homol- /20 ogy between the two viral genomes. /Myr\ DISCUSSION COLOBINAE ERCOPITHECINAE In this paper, we report the isolation and initial characterization COLOBUS TYPE C MACAQUE TYPE C of a new endogenous retrovirus of primates from the Old World ? ~ BABOON TYPE C monkey colobus. This virus has been designated CPC-1 for LANGUR TYPE D ? Colobus polykomos type C virus. CPC-I virus contains reverse FIG. 3. Species oforigins ofthe known endogenous retroviruses transcriptase activity antigenically related to previously isolated of primates. Points of evolutionary divergence between primate mammalian type C viruses and can be reproducibly transmitted families and subfamilies are indicated in millions of years (Myr) and to both human A549 and cat FEF cells. Nucleic acid hybrid- are taken from estimates based principally on the record. ization studies have shown that the CPC-1 genome is present in 50-70 copies in colobus monkey cellular DNA and that partially related sequences are present in a similar copy number langur) and apes (gibbon, orangutan, and possibly humans) (12, in the DNA of other Old World monkeys. CPC-1 is therefore 26). In like fashion, the precise pattern of hybridization of the an endogenous type C virus of the colobus monkey, and ho- CPC-1 (and MAC-1) transcript to primate DNA may also be mologous virogene sequences appear to have been genetically partially explained in terms of the Asian or African origin of preserved in various other Old World monkey genomes. different primate species. Because CPC-1 is derived from an In terms of both nucleic acid sequence homology and anti- African Old World monkey (colobus), the level of hybridization genic crossreactivity, CPC-1 is partially related to MAC-I, the should be somewhat greater to African Old World monkeys, endogenous type C virus isolated from stumptail macaques. A such as baboon, patas, and macaques (which range in virus virtually identical to MAC-I has also been isolated from as well as ), than to a strictly Asian Old World monkey such a Macaca mulatta cell line and designated MMC-1 (9). Both as the langur. Moreover, the level of hybridization to African CPC-1 and MAC-I show no appreciable sequence homology apes such as chimpanzee should be greater than to Asian apes to any of the other previously isolated endogenous primate such as the orangutan and gibbon. These are, in fact, the results retroviruses, including M28 baboon type C virus, OMC-1 owl obtained (Table 1). Thus, hybridization studies with CPC-1 viral monkey type C virus, PO-i-Lu langur type D virus, and M534 transcripts lend further support to the hypothesis that the extent squirrel monkey type D virus. In addition, CPC-1 and MAC-1 of conservation of specific virogene sequences in the genomes appear to have an identical host range in vitro. CPC-1 and of Old World monkeys and apes parallels the Asian or African MAC-1, then, are related to each other but are distinct from all of these primates or their ancestors. of the other classes of endogenous primate retroviruses. CPC-1 It is important to emphasize that the isolation of CPC-1 virus and MAC-I, therefore, represent separate isolates from the same was the result of a single, long-term cocultivation experiment class of endogenous primate type C viruses and, as such, com- and that the culture had remained negative for viral polymerase prise the first example of two analogous retroviruses isolated activity until nearly 8 months had elapsed. Thus, the isolation from primate species separated in by nearly 20 mil- of CPC-1 virus is another example of "low-frequency" virus years. activation in primates (8). Other examples of low-frequency Fig. 3 summarizes the evolutionary relationships of those virus activation among previously isolated primate retroviruses primate species from which endogenous retroviruses have been include MAC-1 stumptail macaque type C virus (8), MMC-1 isolated. As shown, of the six genetically transmitted primate type C virus (9), OMC-1 owl monkey type C viruses isolated to date, the only two viruses related to each other virus (7), and PO-i-Lu langur type D virus (6). These viruses are the type C viruses obtained from the colobus and macaque. were all isolated in single experiments lasting from 7 to 8 Virus isolates homologous to the endogenous viruses of owl months. The isolation of these viruses and of CPC-1 contrasts monkeys, squirrel monkeys, langurs, and baboons have not been strongly with the "high-frequency" activation of the endoge- obtained from distantly related primates. nous baboon type C virus (1-3, 13) and endogenous squirrel CPC-1 and MAC-1 viruses are also similar in their pattern monkey type D virus (4,5). The latter viruses have been isolated of hybridization to the DNA of other primate species. Both repeatedly and rapidly from various tissues and on a wide range CPC-1 and MAC-1 viral transcripts hybridize more to the Old of host cells. Thus, it would now appear that a majority of pri- World monkeys belonging to the Cercopithecinae subfamily mate species are capable only of low-frequency virus activation. than to the langur and more to chimpanzees than to other apes Further isolations of additional endogenous primate retrovi- and humans (Table I and ref. 8). The African Old World ruses-including perhaps those of apes and humans-may monkeys (baboon, mangabey, patas, vervet, and colobus) and therefore require prolonged cocultivation. with unusually apes (chimpanzee and ), show a considerably greater permissive host cells. degree of homology to the African-derived baboon type C vi- We thank Linda Papke, Janis Koci, and Nancy Cope for their expert ruses than do the Asian Old World monkeys (macaque and technical assistance. 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