Type D Primate Retroviruses: a Review1

[CANCER RESEARCH 38, 3123-3139, October 1978] Type D Primate Retroviruses: A Review1

Donald Fine and Gerald Schochetman

Viral Oncology Program, Frederick Cancer Research Center. Frederick, Maryland 21701

Abstract

The prototype virus of the type D retroviruses is the techniques. The retrovirus associated with the induction of Mason-Pfizer monkey virus (MPMV). MPMV was originally mammary cancer in mice, MMTV,2 is morphologically dis isolated from a breast carcinoma of a female rhesus tinct from the type C viruses and is the prototype of the type monkey (an Old World monkey). MPMV is of obvious B virus group (30). A retrovirus group of primate origin, importance in that it is the only retrovirus thus far isolated which has properties similar to the type C and type B from a mammary tumor of a primate and has been shown viruses but is morphologically distinct from them, has been to have transforming potential for primate cells in vitro. designated genus Oncornavirus D (30). The prototype virus Subsequent to the isolation of MPMV, viruses morpholog of this genus is the MPMV. MPMV was originally isolated ically and Â¡mmunologicallyindistinguishable from MPMV from a mammary carcinoma of a female rhesus monkey (an have been isolated from normal placenta and lactating Old World monkey) (19, 60). Because mammary carcinomas mammary glands of other rhesus monkeys in captivity. represent the most prevalent form of neoplasia in humans Recently, viruses morphologically resembling MPMV have and, furthermore, because the incidence of mammary tu been isolated from a langur monkey (another Old World mors in subhuman primates is rare (61, 62, 70, 78), MPMV monkey) and from squirrel monkeys (a New World mon is of obvious potential importance because it is the only key). Based on nucleic acid hybridization studies, the retrovirus thus far isolated from a primate mammary tumor. latter 2 viruses represent endogenous viruses in their Recently, there have been reports that viruses morpho species of origin, whereas MPMV appears to be a horizon logically and immunologically similar to MPMV have been tally transmitted virus containing gene sequences par isolated from certain rhesus monkey and human tissues tially related to the langur monkey isolate. Studies on the and cell lines (6, 8, 10, 44, 49, 55, 57-59, 72, 81, 107-109). immunological relatedness of the type D retroviruses Virus morphologically resembling MPMV have also been have demonstrated interspecies cross-reactivities be isolated from normal tissues of a langur monkey (99) (an tween the major internal and external proteins of the other Old World monkey) and from squirrel monkeys (53, viruses. Furthermore, these viruses also share cross- 99) (a New World monkey). Only the langur isolate shares reactivity of their major external glycoproteins with those significant immunological cross-reactivity and nucleic acid of the type C baboon endogenous virus. These interspe sequence homology with MPMV. The discovery of these 2 cies reactivities can also be demonstrated in natural sera viruses makes it apparent that MPMV is a member of a from both imported and laboratory-bred monkeys. The larger group of viruses of both Old and New World pri demonstration of these interspecies cross-reactivities mates. Furthermore, the large accumulation of information shared by distantly related primate retroviruses provides on MPMV and the new type D primate isolates warrants a a means for detecting determinants that are representa comprehensive review of these viruses. This review will tive of all primate retroviruses presently known and yet to consider the unique characteristics of type D retroviruses be isolated and may provide new assays for detection of as well as their distribution in the primate population and a human retrovirus. their relevance to human cancer.

Introduction History

Over the past 60 years, a considerable amount of infor MPMV. MPMV was first observed and isolated from a mation has been amassed concerning the role of viruses as tissue biopsy performed on an 8-year-old female rhesus etiological agents of animal neoplasia. In particular, those viruses known as RNA tumor viruses or retroviruses have 2 The abbreviations used are: MMTV, mouse mammary tumor virus; been implicated in a variety of cancers in a wide variety of MPMV, Mason-Pfizer monkey virus; CMMT, cocultivated monkey embryo and mammary tumor; PO-1-Lu, langur virus; SMRV, squirrel monkey retro- animal species. Those associated with the induction of virus; RT, reverse transcriptase; A204, human rhabdomyosarcoma; B-GPV, lymphomas, leukemia, or sarcomas have been termed type guinea pig endogenous virus; BaEV, baboon endogenous virus; AMV, avian C viruses based on their morphology as determined by myeloblastosis virus; BLV, bovine leukemia virus; EIAV, equine infectious anemia virus; oligo(dT)-poly(rA), noncovalent copolymer of oligodeoxythy- electron microscopy (14). They have been isolated from midylate and polyriboadenylate; oligo(dG)-poly(rC), noncovalent copolymer several vertebrate classes and are structurally, biochemi of oligodeoxyguanylate and polyribocytidylate; SDS-PAGE, sodium dodecyl cally, and biologically similar, although they are distin sulfate-polyacrylamide gel electrophoresis; gp, p, followed by 2 numbers, glycoprotein and protein, respectively, followed by 2 numbers designating guishable by sensitive immunological and biochemical molecular weight in thousands (e.g., gp70, p27); gp69/71, glycoprotein with a molecular weight range of 69.000 to 71,000; RD114, feline endogenous virus; KC, human glioma; RHFS, rhesis monkey foreskin; RIA, radioimmuno- 1 This work was supported by the Virus Cancer Program, Contract N01- assay; FeLV, feline leukemia virus; cDNA, complementary DNA, SSV, woolly CO-75380, National Cancer Institute, NIH, Bethesda, Md. 20014. monkey virus; GALV, gibbon ape virus; BaLV, baboon leukemia virus; Received April 5, 1978; accepted June 16, 1978. F-MuLV, Friend leukemia virus; R-MuLV, Rauscher leukemia virus.

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monkey (Macaca mulatta). This animal had developed a present in all HeLa cell stocks, as measured by nucleic acid spontaneous tumor (69) in the region of the left mammary hybridization, but are present in those cultures expressing gland after being in a state of continuous estrus for almost MPMV (25). Numerous isolations of MPMV-like viruses from 1 year. The animal was sacrificed approximately 2 months a variety of human cell cultures have been reported; Table after the discovery of the tumor, at which time virus was 1 lists the names and sources of the various isolates. observed in tumor samples by thin-section electron micros Recently, many of these cell lines were shown to possess copy (19, 23). At necropsy the tumor was found to have HeLa cell chromosomal and isoenzyme markers (65, 75). It metastasized into both breasts, the left axilla, flank, and rib is possible that these cell cultures are examples of cross- cage. Secondary tumors were also found that involved a contamination of the original cell cultures with HeLa cells, number of organs including an ovary, the adrenals, pan and it seems reasonable to assume that the virus isolates creas, kidney, liver, stomach, and lymph glands. The pri also represent laboratory contaminants.3 Interestingly, mary tumor histologically resembled a medullary carcinoma these virus isolations occurred in separate laboratories lacking lymphoid stroma (69). Because the tumor did not around the world and, supposedly, none of the research closely resemble conventional breast adenocarcinomas mi groups was conducting research on MPMV. These striking croscopically and because surrounding breast parenchyma observations indicate the widespread distribution of viruses did not present any evidence of developing in situ malignant indistinguishable from MPMV and demonstrate the propen changes, the possibility was raised that the tumor arose as sity of these viruses for infection of human cells. These an intramammary anaplastic lesion (M. Black, personal features dictate the need for extreme caution in studying communication). primate-derived retroviruses with regard to their role in the Since the tumor specimen grew poorly when placed into etiology of human cancers. culture, virus isolation was effected by cocultivation of PO-1-Lu. A new retrovirus resembling MPMV was isolated minced tumor tissues with primary or early-passage monkey from another Old World monkey, Presbytis obscurus, the embryo cell cultures (60). This led to the establishment of a spectacled langur (99). The virus, designated PO-1-Lu, was continuous cell culture system (CMMT) which served as a recovered from cultures of lung tissue cocultivated with bat producer of MPMV. Cultures of CMMT cells transferred for lung cells (TbILu) and could be distinguished from MPMV more than 95 passages consist of cells with distinct rhesus based on differences in host range, antigenic characteris monkey karyotype (74). Although CMMT cultures were tics, and nucleic acid sequence homology. Complete pro- originally maintained by adding nonirradiated rhesus em viral sequences of PO-1-Lu have been found in langur bryo cells to the MPMV-infected cells every 30 days, high monkeys, indicating that the virus is endogenous in this passages of the culture grew without further cocultivation species (12). (74). Virus from the original tumor was successfully trans SMRV. Another retrovirus, SMRV, was isolated from fetal mitted as a cell-free filtrate with the use of low-passage lung tissue of SaimirÃsciureus, a New World monkey (53). monkey embryo cells (60). The isolation was achieved by cocultivation of fetal lung To determine whether MPMV or MPMV-related viruses tissue with canine thymus (Fcf2th) cells. Additional viruses were present in tissues of apparently normal rhesus mon resembling SMRV were isolated from a variety of tissues of keys, Ahmed et al. (6) examined lactating breast tissue, S. sciureus by the cocultivation technique (53, 99). The placentas, and fetuses of monkeys in a closed breeding original isolates derived from squirrel monkeys have been colony at Kensington, Md. These workers were successful designated SMRV (53). A subsequent squirrel monkey virus in isolating 2 new MPMV-related isolates; one, designated isolate has been referred to as M534 (99). Although the X-381, was derived from a lactating mammary gland biopsy, virus contains a Mg2+-preferring RT and is morphologically and the other, designated FTP-1, was derived from placen- similar to MPMV, it shares neither group-specific immuno- tal tissue cocultivated with human NC37 lymphoblastoid logical relatedness nor nucleic acid sequence homology cells. Although MPMV antigens and budding MPMV-like with MPMV. The ability to isolate SMRV from a variety of particles were observed in 3 other mammary biopsy cul organs of more than 1 squirrel monkey (53) and the fact tures, they were lost as a result of simian foamy virus that all tissues of squirrel monkeys contain SMRV proviral contamination (6). sequences (26, 41, 54, 91) demonstrate that SMRV is an The fact that viruses similar to but not identical with endogenous virus of this animal species. A recent report MPMV could be isolated from normal rhesus monkey tissue (100), which indicates that the isolation of endogenous raises the possibility that in the original monkey tumor primate viruses from species other than baboons is difficult, tissue MPMV was present fortuitously and was not etiologi- stresses the importance of the isolation of both SMRV, the cally involved in tumor development. Consistent with this first endogenous virus from a New World primate, and PO- possibility was the fact that virus particles morphologically 1-Lu, the first non-type C endogenous virus from an Old similar to MPMV were also observed in several nontumor- World primate. ous tissues, including the thymus and the lymph nodes of the original tumor-bearing animal (23). Virus Classification A virus thus far indistinguishable from MPMV was also In 1974, Retroviridae was established (30) as a family of isolated from HeLa cell cultures by Bauer ef al. (10, 44, viruses that contain a 60 to 70S single-stranded RNA and an 103). The presence of MPMV in these HeLa cell cultures probably represents a laboratory contamination subsequent 3 For proper referencing we have referred to each virus isolate according to the establishment of the cell line. This conclusion is to the cell designation used in the original isolation although, as stated, based on the fact that MPMV provi ral sequences are not many if not all of the cell cultures are of HeLa cell origin.

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Table 1 Type D primate retrovirus isolates descrip OriginSubhuman tionMPMVX-381FTP-1PO-1-LuSMRVM534J96HEp-2HedescriptionSpontaneoustissue primatesM. mulattaP. tumorof mammary monkeyLactatinga female rhesus afemale mammary gland of monkeyPlacentarhesus rhesusmonkeyLungof a female

obscurisS. lan-gurLungtissue of a spectacled

sciureusHuman monkeyLungtissue of a squirrel monkeyLeukemiaEpidermoidcells of a squirrel

(Homo sapiens)Virus carcinomaCervical 1091010958,1088110910915,59,7249 LaHeLa-ACaOvAODAPTRHDetroitcarcinomaClone carcinomaOvarianof cervical carcinomaAmnionAstrocytomaSpontaneously

transformedrenal cellsBoneembryo 6GraffiHost withlungmarrow of patient carcinomaEmbryonic cells from fetus de rived from female with gener alized portio carcinomaRef.606699539910957,

antigenically specific RNA-dependent DMA polymerase. spaces. The latter particle contained both an outer unit termed RT. The various members of the family Retroviridae membrane and a distinct membrane binding the nucleoid. are subdivided into 6 genera designated A through F, a MPMV, which was subsequently propagated in human classification based largely on morphological distinctions lymphoblastoid (NC37) (60), A204 (48, 81), or rhesus mon set forth by Bernhard (14). Type A particles, which are 60 to key (MA-101, CMMT) (60, 64, 67) cell cultures, has exhibited 90 nm in diameter, consist of 2 forms, intracytoplasmic and particle types with these basic morphologies. intracisternal. Both types are double-shelled and have an Prior to the establishment of the family Retroviridae, electron-lucent center. The role of the intracisternal A MPMV was considered to be morphologically similar to particle (which constitutes genus Cisternavirus A) is un MMTV, the prototype type B virus (23, 64, 67). Chopra known; however, the intracytoplasmic A particles are con and Mason (23) first correlated MPMV with MMTV based on sidered to be precursors to the type B particles (30). their observations that extracellular virions consisted Intracytoplasmic A particles (Fig. 1a) and type B particles largely of enveloped particles containing an eccentric (Fig. 1, b and c) are 2 morphological forms associated with dense nucleoid and that virus development occurred intra- members of the genus Oncornavirus B, generally termed cytoplasmically in the form of "A" particles (Fig. 1g), the type B virus. Type B particles bud at the cell membrane thought to be the precursor of the type B particle. Although (Fig. 1b) with a complete nucleoid to form extracellular Chopra and Mason (23) pointed out that the extracellular particles, the mature form of which has an eccentric nu form of MPMV resembles the type C viruses (i.e., character cleoid and prominent surface spikes on its outer envelope istically devoid of surface spikes), Kramarsky ef al. (64) (Fig. 1c). The members of the genus Oncornavirus C, more found MPMV to be in the size range of MMTV (125 to 130 commonly termed type C viruses, include those viruses of nm), larger than the typical avian and murine type C viruses various animal species that have no intracytoplasmic mor (110 nm) previously examined. The latter investigators also phologically identifiable form (Fig. id) but that bud with a distinguished MPMV from MMTV on the basis of the size of crescent-shaped nucleoid at the cell membrane (Fig. 1e). the intracytoplasmic "A" particles (diameters of 90 and 75 The extracellular form of the virus contains a central nu nm, respectively) and the presence of knobs on the virion cleoid and lacks surface projections or spikes (Fig. 1f ). In envelope of MPMV, in contrast to the longer spikes on the their description of the virus particles found in the rhesus envelope of MMTV. On the basis of these distinguishing breast tumor from which MPMV was first isolated, Chopra morphological characteristics, MPMV was placed in the et al. (19, 21, 23) observed both intracellular (Fig. 1, g and genus Oncornavirus D (Fig. 1/). MPMV was also distin h) and extracellular (Fig. 1;) particle types. The intracellular guished from the foamy viruses of monkeys and chimpan particles were ring shaped, measured 60 to 95 nm in zees (22), which are grouped in the genus Spumavirus F, in diameter, and appeared near the plasma membrane. The that the latter contain an electron-lucent nucleoid and extracellular or "mature" particles, measuring 100 to 120 prominent surface spikes ranging from 5 to 10 nm in length. nm in diameter, contained an electron-dense nucleoid and The rhesus monkey isolates X-381 and FTP-1 (6) and the were free as well as attached to membrane in intracellular langur isolate PO-1-Lu (99) exhibit virus maturation identi-

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. D. Fine and G. Schochetman cal with that of MPMV. The virus particles observed in RNA subunits may be linked by differentially stable hydro certain HeLa cell cultures (44) and in a cell culture derived gen-bonded RNA regions (94). Complete heat dissociation from human brain (55) were also indistinguishable from of MPMV 70S RNA also yielded 3 low-molecular-weight MPMV. Virus particles morphologically resembling MPMV RNA species of 2.5 x 104, 3.5 x 104, and 1 x 10s daltons, have been observed in other cell cultures derived from corresponding to approximate sedimentation coefficients human tumors (49, 72, 81). of 4, 4.5, and 7S, respectively (94). These small RNA's are Although SMRV was originally reported to resemble similar to those found in other retroviruses. MPMV morphologically (53), subsequent comparative elec As reported for other retroviruses, MPMV contains a tron microscopy studies (48) emphasizing thin-section and polyadenylic acid sequence of approximately 200 nucleo- critical-point drying techniques showed that the extracellu tides covalently linked to the viral RNA (47). Although not lar mature forms of the 2 viruses are distinguishable. SMRV reported these sequences presumably are located at the 3' is generally round with a central electron-dense nucleoid, end of the molecule. whereas MPMV has an irregularly shaped envelope enclos SMRV (26, 41, 91) and the MPMV-like isolates from AO ing a tubular or barrel-shaped nucleoid. (16, 81), J96 (81), HEp-2 (109), and Detroit-6 (16, 72) cells Additional properties used to characterize retroviruses also contain 60 to 70S RNA. However, relatively little has include buoyant density analysis in different media and been reported about the RNA structure of these and the divalent cation preference for the viral RT. MPMV (93), as other MPMV isolates. well as X-381, FTP-1,4 and SMRV (92), exhibit a buoyant Viral RT. The RT of retroviruses convertsthe viral genetic density of 1.17 g/ml in sucrose and 1.21 g/ml in CsCI. This information, which is in the form of RNA, into a DNA property is also shared by the type B viruses, MMTV (89) molecule which is subsequently integrated into the host and B-GPV (71), and at least 1 member of the primate type cell DNA and allows for the vertical transmission of the viral C viruses, BaEV.4 In contrast the murine type C viruses information in an unexpressed form as an integral part of exhibit a single buoyant density of 1.16 g/ml in both the cell genome. sucrose and CsCI (89). MPMV has been shown to have a virion-associated RT MPMV (1, 33, 105), X-381 (105), FTP-1, SMRV (26, 54, 92), (1). The enzyme has been purified by a variety of techniques PO-1-Lu (99), and retroviruses isolated from HeLa (15, 103), including ion-exchange chromatography followed by gel HEp-2 (15), and AO (15) cells exhibit a strong preference for filtration (1, 56) and ion-exchange chromatography fol Mg2* as the divalent cation for viral RT activity, whereas all lowed by affinity chromatography on polyribocytidylic acid- members of the murine and primate type C viruses have agarose (29). The molecular weight of the MPMV RT has been shown to have a cation preference for Mn2+ (33, 95). been reported to be 110,000 (1). A more recent estimate Other viruses having RT's with a preference for Mg2* (29) placed the molecular weight at about 80,000, interme include those of mouse, MMTV (33); avian, AMV (50); diate in size to the mammalian type C (m.w. 70,000) and bovine, BLV (46); and equine, EIAV (17) origin. type B (m.w. 100,000) viral RT's (1, 56, 73). This latter Although various members of Oncornavirus B and C estimate is in good agreement with the molecular weight determinations for partially purified RT's from the SMRV possess 1 or more of the above morphological, biophysical, and biochemical properties, only MPMV, SMRV, PO-1-Lu, (m.w. 80,000) (26) and PO-1-Lu (m.w. 85,000 to 90,000) (99). X-381, FTP-1, and the HeLa isolate possess all of the Both MPMV virion-associated and purified RT have been following properties: (a) budding from the cell membrane shown to prefer Mg24 as the divalent cation in an endoge with a complete nucleoid; (b) intracytoplasmic A particles; nous reaction in addition to reactions utilizing the synthetic (c) extracellular particles having a central nucleoid and templates oligo(dT)-poly(rA) and oligo(dG)-poly(rC) (29). lacking prominent surface projections; (d) buoyant density With the synthetic template oligo(dG)-poly(rC) or of 1.16 g/ml in sucrose and 1.21 g/ml in CsCI; and (e) Mg2*- oligo(dT)-poly(rA), PO-1-Lu (99), SMRV (26, 53, 92), the preferring viral RT. The above properties are summarized in virus isolates from HeLa (103), and HEp-2 (58) cells were Table 2 for representative retroviruses. On the basis of this also shown to prefer Mg2" as the divalent cation. The HeLa information, we suggest that those viruses that possess all cell isolate has also been reported to possess RNase H of the properties in common with MPMV be placed in the activity (103). genus Oncornavirus D. Structural Proteins. MPMV has been shown to contain 6 major proteins with molecular weights of 68,000 to 70,000; Virus Structure 27,000; 20,000; 14,000; 12,000; and 10,000 as determined by SDS-PAGE (93, 101), similar to those reported for type C Like other retroviruses, MPMV contains a 60 to 70S RNA viruses (9). On the basis of glucosamine labeling, the 68,000 (52, 67, 94), which has a molecular weight of approximately to 70,000 (gp70)-dalton protein is the major virion glycopro- 8 x 106daltons when analyzed by electrophoresis in poly- tein and the 20,000 (gp20)-dalton protein is a minor glyco- acrylamide-agarose gels (94). MPMV 60 to 70S RNA's from protein (93). This is demonstrated by the fact that gp70 is short-term (2-hr) harvests, when denatured by heat (80Â°,2.5 labeled approximately 15 times better than is gp20 on a min) or formamide (40%), yielded subunit structures of 2.8 molar basis (93). Surface iodination of intact MPMV cata x 106daltons (94). Treatment of viral 70S RNA with limiting lyzed by the enzyme lactoperoxidase demonstrated that amounts of formamide resulted in a stepwise dissociation gp70 was the major external virion protein (93). gp20 has to intermediate RNA structures, suggesting that the MPMV been reported to have a molecular weight of 15,000 to 17,000 by gel filtration in 6 M guanidine hydrochloride (93, ' G. Schochetman, unpublished data. 101). This difference in molecular weight is probably due to

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Table 2 Properties of representative members of genera Oncornavirus B, C, and D density NucleoidType (g/ml)Sucrose1.171.16

A extracellular projec cation particlesbudding+ At virusEccentric, tionsLong chloride1.21 preferenceMg2+ B Complete (MMTV) denseCentricCentric, (spikes) Type C Incomplete ShortShort 1.16 Mn2+ (MuLV) Type D + CompleteIn 1.17Cesium 1.21RT Mg2+ (MPMV)plasmic denseSurface (knobs)Buoyant the anomalous migration of glycoproteins in SDS-PAGE. mined by SDS-PAGE. Four major polypeptides with molec That these 6 proteins are virus-coded and not host cellular weights of 40,000, 20,000, 14,000, and 8,000 were derived components is indicated by observations that none resolved in virus propagated in human, mink, and canine of the proteins was selectively lost during extensive purifi cells (92). This result indicates that these 4 proteins are cation of the virus and that all were present in the same virus coded and that the 40,000-dalton component is the relative proportions, regardless of the cell type from which major nonglycosylated protein of SMRV. A subsequent the virus originated (93). In addition p27 (the major nongly- study with agarose gel filtration under denaturing condi coprotein) was found only in MPMV-infected cells and was tions (31) estimated the molecular weight of this protein to antigenically indistinguishable from virus grown in 3 differ be approximately 35,000; thus, this protein will be referred ent primate cell cultures (90, 93). These results further to as p35. SMRV propagated in mink cells also contained a indicate that p27 is induced after virus infection and thus 73,000-dalton protein, presumably similar to the gp73 com represents a virus-coded protein. Two additional proteins, ponent of SMRV grown in A204 cells. In contrast SMRV gp48 and gp36, of uncertain origin, were found only in virus from canine cells contained a 100,000-dalton polypeptide grown in NC37 pells (93). Numerous type C viruses propa instead of the 73,000-dalton component (92). gated in NC37 cells contain proteins of similar molecular weight (M. Ahmed, personal communication), which may Biological Properties indicate that these proteins are host derived. However, additional studies are necessary to verify their origin. The Infectivity. Cocultivation of cells in culture has provided possibility exists that 1 or both of these proteins represent an important tool for the isolation of retroviruses, particu a differentially glycosylated form of the major glycoprotein larly of the type D family (6, 53, 60, 100). Although MPMV gp68. An example of this phenomenon has been reported was first isolated by cocultivation, it was also obtained by for murine type C viruses, in which an incompletely glyco direct infection of rhesus monkey embryo cells with tissue sylated form of the major envelope glycoprotein gp69/71 culture fluids from the original rhesus monkey breast tumor exists as a molecule with an approximate molecular weight cell culture (60). This was the-first evidence that MPMV was of 45,000(gp45)(68). infectious for primate cells and could replicate in vitro. The polypeptides of the 2 MPMV-related rhesus monkey Subsequent studies (Table 3) established that the host isolates X-381 and FTP-1 have also been examined and range of MPMV is primarily restricted to primate cell cul shown to be identical with those of MPMV. The FTP-1 tures, including those of human origin, although productive isolate, propagated in NC37 cells, also exhibited 2 addi infections have been accomplished recently in cell cultures tional glycoproteins, gp48 and gp36, as was observed for of horse epidermis (CCL57) (D. Fine, unpublished data) and MPMV grown in NC37 cells (93). The HeLa isolate contained Aleutian mink lung (CCL64) (28). However, the horse epi several low-molecular-weight proteins with molecular dermis cells produce approximately 10-fold less virus than weights between 12,000 and 28,000. Also present were 2 do infected human cell cultures. glycoproteins with molecular weights of 60,000 and 80,000 PO-1-Lu, like MPMV, also exhibits a restricted host range (10). Although several of these proteins correspond to the (Table 3) and replicates best in human (A204) and bat major proteins of MPMV, a more detailed characterization (TbILu) cell cultures (99). In contrast, SMRV has a broad of this virus is required to clarify this point. host range (Table 3) and can successfully replicate in The protein composition of MPMV and related isolates is human, rhesus monkey, and a variety of nonprimate cells clearly different from that of BaEV, a type C virus of Old (53, 92, 99). PO-1-Lu and SMRV share the common property World monkeys (97). At present the structural proteins of of not replicating in cells of their respective host of origin the PO-1-Lu isolate have not been examined. It would be of (53, 99) and thus may represent examples of xenotropic considerable interest to determine whether PO-1-Lu, which primate retroviruses. is morphologically indistinguishable from but only partially Although infection of permissive cell lines with MPMV related antigenically to MPMV, contains structural proteins generally has no accompanying cytopathology (11, 60, 76), either similar to or distinct from MPMV and BaEV. infection of rhesus monkey and human foreskin cultures The polypeptide composition of SMRV, the endogenous (38, 40) and rhesus monkey embryonic lung cultures (Ref. virus of squirrel monkeys, was clearly distinct from that of 38; E. Hunter, personal communication) resulted in the MPMV and MMTV (the prototype type B virus) as deter formation of multinucleated foci or syncytia in the cell

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Table 3 Hosf range for type D retroviruses Virus Cell line designation and tissue Species origin MPMV PO-1-Lu SMRV Primate Old World monkeys Rhesus (M. mulatta) ND" (mixed embryo) + (60) MA-101 (embryonic lung) + (60, 67) Rhmk (embryonic kidney) + "(7) RHFS (embryonic foreskin) + (3, 7, 38) + (53) DBS-fRhL-1 (lung) + (99) (99) + (99) African green (Cer- Vero (kidney) + (60) copithecus aethiops sabaeus) Baboon (Papio cy- LE-10 (leukocytes) -(60) nocephalus) BFS (fetal skin) -(53)

New World monkeys Squirrel (S. sci- SqMLu (lung) -(53) ureus) Marmoset (Sa- MoLv (lung) guinus sp.) MAK (kidney) -(53) Owl (/lofes trivirga- OMK (kidney) -(53) tus)

Howler (Alouatta HMS (skin) -(53) belzebul) Apes Chimpanzee (Pan ND(lung) + (60, 67) troglodytes) SFRE:CL-1 (lung) + (60) + (53) LE-11 (leukocytes) -(60) Human (H. sapiens) A204 (rhabdomyosarcoma) + (7, 11,81,99) (99) + (53, 92, 99) RD (osteosarcoma) + HOS (osteosarcoma) KC (glioma) -J4.86) HeLa (cervical carcinoma) AY603 (mammary adenocarcinoma) MCF-7 (mammary adenocarcinoma) NC37 (lymphoblast) -^(7,24,60) HBL-100 (mammary epithe lium) HE-37 (mixed embryo) + (60) HE-40 (mixed embryo) + (60) HSO410 (fetal fibroblast) + (53) HuFS (foreskin) + (38) HEK (embryonic kidney) + (4, 7, 22, 48) WI38 (diploid) + (7, 86) IMR-90 (diploid)

Nonprimate Horse (Equus cabal- E. derm (dermis) lus) vison)DogMink (Mustela (lung)MvlLu (7,28)-(99)-(99)-(99)- (53,92)+ (64J1)(lung)Fcf2th (99)+ famili-aries)Cat(Canis (thymus)MDCK (53, 92,99)+

(kidney)FEC (53)-(99)+ (Felisdomesticus)Bat (embryo)TbILu brasilien-sis)Hamster(Tonatia (lung)BHK (7,99)- (99)-(99)+ (99)+

(Mesocrice-tus (kidney)SIRC (7,60)-(99)-(7)-(7)- auratus)Rabbit (Oryctolaguscuniculus)Mouse(cornea)LLC-RK, (99) (kidney)ND (lung)JLS-V9 (M.musculus)Rat marrow)NRK(bone (7,60)-(7)-(99)-(99)-(99)+ (Rattusnorvegi-cus)MvlLu (kidney)+

" ND, no designation cited. * D. Fine, C. Clarke, and L. Arthur, unpublished data. 3128

monolayer. Syncytia with similar morphologies are induced dependence, which enables them to grow in semisolid by human and feline syncytia-forming viruses (51, 66) and agar. In addition MPMV-transformed RHFS and horse epi RD114 (85). These syncytia were clearly distinguishable dermis cultures exhibit an altered morphology, loss of from those caused by simian foamy viruses by size (number contact inhibition of movement, and chromosomal abnor of nuclei per syncytium), time of appearance, and vacuoli- malities (3, 7,40,83). zation of the culture monolayer (38). Development of syn MPMV-transformed RHFS and horse epidermis cells cytia also occurs when KC cells (a Rous sarcoma virus- chronically produced virus that was indistinguishable from transformed human glioma cell line) are infected with the virus used in the original inoculum (CMMT-MPMV). This MPMV, BaEV, and RD114 (2, 4, 85, 86). In RHFS and KC conclusion was based on the SDS-PAGE pattern of viral proteins (93) and complete identity of viral p27's (90) and cells, the number of syncytia formed correlates with virus dose and provides the basis for a virus infectivity assay (4, gp70's (G. Schochetman, D. Fine, S. Devare, and J. Ste- phenson, unpublished data) in homologous MPMV RIA's. 38, 86). Syncytia formation occurs within 24 hr after infec tion in both cell lines (38, 86), is neutralized by MPMV The ability of MPMV-transformed cells to grow in semi- antiserum (40), and is enhanced by Polybrene or DEAE- solid agar offers a means for determining frequency of dextran treatment prior to infection. Syncytia persist in the transformation and for isolation of transformed cell clones. MPMV-infected cultures for 2 to 4 weeks and then disap Transformation in MPMV-infected RHFS cell cultures oc pear, resulting in chronically infected cultures that are curred at a low frequency (approximately 10 5) and was resistant to superinfection with MPMV (40, 86). SMRV was dependent on multiplicity of input virus (3, 7, 83). Unin- initially reported to induce syncytia on KC cells (53). How fected RHFS cultures were fibroblastic and exhibited con ever, this phenomenon is not reproducible, although the tact inhibition of movement, whereas sublines established copresence of SMRV with MPMV in the KC cells enhances from colonies of MPMV-transformed RHFS cells had a MPMV-induced syncytia (D. Fine, unpublished data). As polygonal epithelioid phenotype, displayed loss of contact yet, PO-1-Lu has not been reported to induce syncytia in KC inhibition of movement, and contained foci of clumped cells. cells and syncytia. These transformed sublines grew 20 to A recent study (E. Hunter, personal communication) dem 90 times better in serum factor-free medium than did onstrated that UV irradiation of MPMV inhibited viral-in uninfected rhesus foreskin cells (83). duced syncytia in rhesus monkey embryonic lung cells and MPMV-transformed RHFS cells with epithelial morphol that the inactivation followed single-hit kinetics. This result ogy had a high plating efficiency in soft agar and exhibited excludes a direct effect on the gp70 of the virus. The chromosomal abnormalities characterized by polyploidy inactivation of gp70 would be expected to follow multiple- and structural rearrangements, including chromosomal hit kinetics. However, preexposure of KC cells to the gp70 breaks (7, 40). A second cell type, isolated from soft-agar of RD114 blocked syncytia formation by both MPMV and colonies of MPMV-infected RHFS cells in the studies of RD114 (84). If the viral genome were the UV irradiation Ahmed ef al. (3, 7), had a fibroblastic morphology similar to target, it would suggest that a functional viral genome was that of uninfected RHFS cells. The cultures showed lack of necessary for syncytia formation and would explain the contact inhibition, a normal rhesus chromosomal comple relatively long time (approximately 24 hr) required for ment, and a lower plating efficiency in soft agar. Both MPMV-induced syncytia formation. Therefore, the inhibi epithelioid and fibroblastic transformed cell types produced tion of syncytia formation by preexposure of KC cells to the intracellular MPMV antigens and virus particles, but the gp70of RD114 may have resulted from saturation of specific fibroblastic sublines generally produced >2 log]0 50% tis receptor sites shared by MPMV and RD114, thus preventing sue culture-infectious doses/0.5 ml less infectious virus attachment and penetration of the virus. This is important than did the epithelioid sublines. in light of the recent demonstration of interspecies cross- One fibroblastic subline of MPMV-transformed RHFS reactivity between the gp70's of MPMV and RD114 (96). cells was found to express MPMV antigen and virus parti MPMV infections occur in the absence of syncytia forma cles that contained p27, but the virus recovered from the tion in cell lines such as A204 (11), human osteosarcoma culture fluid lacked syncytia-forming capacity in KC cells (Table 3), and SV40-transformed WI-38 (86). In A204 cul (7). Negative-stain electron microscopy showed the mor tures, which are highly permissive for MPMV, newly synthe phology of the virus particles from this subline to be sized virus is detected within 4 days after infection at input distinguishable from KC-positive MPMV produced by other multiplicities significantly greater than 1 virus particle/cell. transformed sublines of RHFS cells (7). KC-positive MPMV At lower input multiplicities (less than 1 virus particle/cell), possessed tails and condensed nucleoids, whereas KC- MPMV infections occur that are inapparent until the cul negative MPMV lacked tails and contained a doughnut- tures have been serially passaged several times. Produc shaped nucleoid. tively infected A204 cultures carried over 30 passages after In Vivo Studies. Attempts to induce tumors by inoculation infection continue to express infectious virus into the extra of MPMV into rhesus monkeys (37, 39), cynomolgus mon cellular fluid. MPMV production in the same infected cul keys, squirrel monkeys, and marmosets have been unsuc tures remains constant based on RT, MPMV p27, and virus cessful (L. Wolfe, personal communication). This failure particle count determinations (11). may be a result of 1 or more of the following: (a) a re In Vitro Transformation. MPMV has been shown to pos quirement for a long latent period not yet reached in the sess in vitro transforming capacity for rhesus monkey, inoculated animals; (b) the original virus inocula contained human, and horse cell cultures (3, 7, 40, 83). One charac little or no transforming virus; (c) MPMV itself is not teristic of these transformed cells is their loss of anchorage oncogenic but requires a helper virus or cocarcinogen; (d)

OCTOBER 1978 3129

MPMV is not oncogenic at all; or (e) effective immune may thus exhibit alternate clinical manifestations such as surveillance by the inoculated host. Consistent with the last those associated with other retroviruses. FeLV, for exam possibility is the ability to detect high titers of both neutral ple, replicates in newborn cats and causes thymic atrophy izing and precipitating antibodies against the whole virus in these animals (35). A runting disease characterized by and several viral structural proteins in inoculated rhesus weight loss and thymic atrophy has been reported in young and cynomolgus monkeys (31). Despite the inability of mice inoculated with Friend leukemia virus (98). Further MPMV to induce tumors, many of the inoculated rhesus more, inoculation of feral mice with a type C viruj indige monkeys became persistently infected, and virus could be nous to these mice induces a high frequency of either hind readily recovered from peripheral lymph nodes (37), saliva, leg paralysis or lymphoma (43). and exfoliated cells in milk (A. Bogden, personal commu Although no oncogenic potential has been demonstrated nication). Maternal transmission of virus via milk in rhesus for purified MPMV, inoculation of MPMV-transformed RHFS monkeys has been observed, providing evidence for at least cells into newborn rhesus monkeys did induce palpable 1 mode of horizontal transmission. nodules of donor cell origin at the sites of inoculation. Although rhesus monkeys neonatally inoculated with Microscopic examination of the nodules revealed a circum MPMV or MPMV-infected cells did not develop tumors, scribed area of proliferating undifferentiated or epithelial many developed severe lymphadenopathy, weight loss, and cells resembling those of the inocula. These cells were thymic atrophy (39). The consistent observation of thymic surrounded by an infiltration of eosinophils, lymphocytes, involution in the inoculated animals during the first several and histiocytes, and there was fibroblastic activity around weeks of age suggests that the thymus may serve as a the periphery. Pretreatment of inoculated monkeys with target organ for MPMV infection in infant monkeys. The rhesus antilymphocyte serum enhanced both nodule size enhanced regression of the thymus may partially explain and duration of appearance (40). The nodules regressed why a large proportion of the inoculated animals suc within 2 to 3 weeks, after which the inoculated animals cumbed to secondary viral or bacterial pneumonia and elicited high-titered MPMV-neutralizing and -precipitating enteritis. Postmortem examination of these animals re antibody (31). Expiant cultures derived from biopsied nod vealed the presence of MPMV in thymus, lymph nodes, ules released infectious MPMV and induced nodules when blood, brain, spleen, kidneys, and bone marrow based on reinoculated into other monkeys (40). Inoculation of MPMV- electron microscopy, induction of syncytia in indicator cell transformed RHFS cells into newborn rats and adult cultures, and immunofluorescence assays with MPMV anti- athymic nude mice (7, 39) also produced small nodules at serum (39). Three characteristic hematological patterns the site of inoculation, most of which regressed within were observed among the inoculated monkeys. One group several weeks after inoculation. MPMV-transformed RHFS showed no hematological aberrations, and less than one- cells resembled chemically transformed human embryo and third of the animals in this group survived. The remainder foreskin cells in their ability to grow in soft agar and their died with symptoms indicative of runting or undue suscep inability to produce malignant tumors derived from donor tibility to facultative pathogens. In these animals MPMV cells when inoculated into athymic nude mice (42). How yielded symptoms indicative of a slow virus infection. The ever, several athymic nude mice that had been inoculated second group exhibited basophilia with or without anemia, with MPMV-transformed RHFS cells did develop invasive and none of these animals survived. The third group devel fibrosarcomas at the site of inoculation. Cytogenetic analy oped neutropenia and/or anemia and most survived (39). In sis of cell cultures derived from the respective fibrosarco these monkeys MPMV yielded symptoms indicative of an mas showed the tumors to possess only mouse karyotype acute virus infection, eliciting hematological responses (39). Virus from these cell cultures did not induce tumors in such as development of both MPMV-neutralizing and high- nude athymic mice, although the mice did elicit high-titered titered precipitating antibodies (31). The precipitating anti precipitating antibodies to both MMTV and MPMV. How bodies were directed against both envelope and internal ever, the cell cultures themselves were highly oncogenic (MPMV p27) antigens and persisted throughout the life of and produced fibrosarcomas in nude athymic, C3H, and the animals suggesting that MPMV existed as a persistent C3Hf mice within 2 weeks. infection in these animals. Antibody-positive animals gen erally remained clinically asymptomatic throughout adult life and showed no great variations in hematological pat Viral Nucleic Acid Sequence Homologies terns when compared with standards for hematological data for control animals. Several MPMV-inoculated rhesus Two techniques of molecular hybridization have been monkeys of the third group succumbed several years after widely utilized in examining the interviral relatedness of the inoculation to neurological syndromes characterized by type D viruses. The first uses a radioactive DNA probe loss of motor reflexes and limb paralysis (D. Fine and W. (cDNA) that is complementary to the viral RNA, and the Loeb, unpublished data; A. Bogden, personal communica second uses radioactive viral RNA as the probe. In the first tion). Postmortem examination of one of these monkeys technique sequence homology between different viruses is revealed marked lymphadenopathy, and syncytia-inducing measured by the degree of hybridization between the cDNA virus was recovered from both lymph nodes and brain probe and the RNA of other viruses. The second method tissues. Furthermore, a newborn rhesus monkey inoculated involves hybridization of radioactive viral 70S RNA to excess with a suspension of brain tissue from this animal devel DNA from viral-infected cells that contain complementary oped a high-titered antibody to MPMV but showed no viral sequences. With this technique sequence homology is measured by the extent to which other viral RNA's can unusual clinical manifestations. Acute MPMV infections

3130 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Type D Primate Retroviruses: A Review compete for the ability of the radioactive RNA probe to Table 4 Natural distribution of type D retrovirus proviral sequences in the hybridize to the cellular DNA. DNA's of various animal species" According to the competitive hybridization technique with radioactive MPMV 70S RNA (25), MPMV was indistin MPMVÂ»PrimatesOldSpecies guishable from X-381, AO, and the HeLa isolate of Bauer (10) and only partially related (approximately 30%) to the WorldmonkeysColobinaeLangur endogenous langur virus, PO-1-Lu (12). No sequence homology was observed between MPMV and the RNA's of cColobus + + + +++ +CercopithecinaeBaboon + ++++++++â€”-â€”--NTNTNTNTNTNTNTSMRVNTNTNTNTâ€”NTNT-â€”-â€”+ SSV, MMTV, murine leukemia virus, B-GPV, AMV (25), and SMRV(26,41,91). +Mangabey With the use of virtually complete [3H]cDNA transcripts of +Macaques PO-1-Lu, approximately 22% nucleic acid sequence homol- +Celebus +Quenon apes ogy was detected with MPMV 70S RNA (12). In reciprocal +Patas experiments, MPMV [3H]DNA transcripts hybridized approx +ApesGibbonChimpanzeeManNew imately 30% to the PO-1-Lu genome (12). In the same studies the PO-1-Lu genome was reported to be distinct from other retroviruses, including GALV, SSV, and the endogenous viruses of Mus cervicolor (M432, CERV CI, and WorldmonkeysSquirrel CERV Cil), Mus musculus (BALB/c xenotropic), and pig monkeyWoolly + ++-â€”â€”NTNT--â€” (CCL33) origin. A low degree of hybridization (4 to 5%) was monkeyCapuchin seen with the RNA of an endogenous baboon virus (M28), NTMarmosetHowlerCebusSaki RD114, and the squirrel monkey virus isolate, M534 (12). The significance of this low level of hybridization is unclear because it is not known whether it represents specific NTSpider sequence homology or higher background hybridization NTOwl monkey values. No sequence homology has been observed between monkeyPo-1-Lu+ SMRV, MPMV, and AMV. These results support the conclu Nonprimates sion that PO-1-Lu is a new viral isolate of Old World Cow monkeys that shares significant sequence homology with Rabbit NT MPMV. Hippopotamus NT On the basis of direct hybridization of [3H]SMRV 70S RNA Sheep NT NT Pig NT NT to the DNA of MPMV-producing cells and competition Dog NT hybridization with [3H]SMRV 70S RNA, no sequence homol Cat NT ogy was observed between SMRV, MPMV, and AMV (26, 41, Mouse NT 91). Direct hybridizations with the use of cDNA of SMRV to Rat NT NT RNA's of MPMV, baboon leukemia virus, SSV, and GALV Chicken NT NT Hamster NT NT also revealed no sequence homology with SMRV (54). Guinea pig NT NT Natural Distribution of Viral Nucleic Acid Sequences. By " Data tabulated from Refs. 12, 25, 26, 34, 41, and 91. definition endogenous virogenes are those sets of gene * Includes X381, FTP-1, and HeLa isolates (indistinguishable sequences that form an integral part of the chromosomal from MPMV based on hybridization and immunoassay criteria). c + + + +, complete homology, + + +, high degree of homology; DNA and that can, under certain conditions, code for the + + , partial homology; +, low homology; -, no homology; NT, not production of a retrovirus. A species of origin has been tested. reported for essentially all type B and type C endogenous viral gene sequences in 1 or more animal hosts. MPMV, although originally isolated from a rhesus monkey (M. thecinae (32 to 45% versus 13 to 16%, respectively) (12). mulatta), until recently remained a virus in search of a The C0f,/2's (20 to 50 mol x sec/liter) for both subfamilies species containing representative endogenous viral gene indicate that these sequences were present in multiple sequences. With direct hybridization of radioactive MPMV copies (20 to 50) as compared to a C,,f,/2 of 900 mol x sec/ 70S RNA to DNA of various animal species, it has been liter in MPMV-infected A204 cells (12). The presence of shown that a portion (approximately 20%) of the MPMV multiple copies has been considered a good criterion for genome was present in the cellular DNA of several Old demonstrating endogenous sequences in host cell DNA, in World monkeys of the subfamily Cercopithecinae (34). No this case endogenous sequences in Old World monkeys. sequence homology was observed between MPMV and the Because the highest final extent of homology (45%) was cellular DNA's of New World monkeys, apes (including obtained with the spectacled langur (P. obscuris) DNA as man), and several nonprimates (Ref. 34; Table 4). This compared with colobus (Colobus guereza) DNA (32%), it observation has been confirmed and extended with the use has been suggested that MPMV was derived from an endog of essentially complete cDNA transcripts of the MPMV enous virus of one of the Asian rather than African genera genome (12). The data obtained with the use of cDNA and, perhaps, from the spectacled langur specifically (12). probes show that MPMV exhibits considerably more nucleic Essentially, full-length cDNA probes of PO-1-Lu hybrid acid sequence homology to the cellular DNA of members of ized completely to DNA of a variety of spectacled langur the Colobinae subfamily than to members of the Cercopi- monkey tissues with a C0f,/2 of approximately 50, consistent

OCTOBER 1978 3131

with the presence of multiple copies (20 to 40 per haploid proteins. On the basis of these assays, MPMV exhibited no genome) of the PO-1-Lu genome (12). The presence of cross-reactivity with type B and type C retroviruses of multiple copies of viral sequences in langur cellular DMA primate (SSV, GALV, BaEV), feline (RD114, FeLV), rodent indicates that PO-1-Lu is an endogenous virus of spectacled (murine leukemia virus, MMTV, B-GPV, pig virus, CCL38), langur monkeys. The same probe hybridizes 75% to Pres- and avian (AMV, RAV-2) origins. In addition, no cross- bytis senex (purple-faced langur) and 59% to C. guereza, a reactivity was observed between MPMV and members of member of the Colobinae subfamily, and from 12 to 15% to the genera Lentavirus (including Visna and maedi virus) the cellular DNA's of 10 different species belonging to 6 and Spumavirus (including the simian syncytial or so-called genera of the Cercopithecinae subfamily (12). The C0f1/2's foamy virus) (see Ref. 30 for retrovirus classification). Nor for these sequences also indicate their presence in multiple does MPMV cross-react with BLV and EIAV, 2 as yet copies. No sequence homology was observed between the unclassified retroviruses. In contrast, the X-381, FTP-1, and PO-1-Lu cDNA probe and the cellular DNA's of New World AO isolates are immunologically indistinguishable from monkeys, apes, including man, and several nonprimates MPMV based on p27 RIA's (Table 5). X-381 and FTP-1 are (12). also indistinguishable from MPMV in a homologous RIA for Using direct hybridization radioactive SMRV 70S RNA MPMV gp70 (Table 5). With the less sensitive technique of hybridized significantly (52 to 77%) to the DNA's of a variety immunodiffusion, the HeLa cell isolate (10) was shown to of squirrel monkey tissues (26, 41, 91). The kinetics of have cross-reactivity with MPMV but not with F-MuLV, hybridization yielded C0f,/2'sof 120 to 550 (26, 91), consist FeLV, and MMTV. The cross-reactivity between MPMV and ent with the presence of the SMRV genome in multiple HeLa virus was supported by use of indirect immunoferritin copies. The presence of multiple copies of viral sequences labeling in which antisera raised against the respective in squirrel monkey cellular DNA indicates that this virus is viruses revealed cross-reacting antigenic specificities on endogenous to S. sciureus. No sequence homology was the surfaces of both HeLa virus and MPMV. Cross-absorp observed between the SMRV 70S RNA probes and the tion and end-point dilution techniques also indicated the cellular DNA's of other New World monkeys, Old World presence of similar if not identical envelope antigens (45). monkeys, apes (including man), and several nonprimates Similarly, with the use of RIA's for MPMV p27, the isolate from J-96 cells has been reported by 2 laboratories to SMRV(91). Similar (13, 54).' results In contrast were reported to the with wide cDNA distribution probes of possess an antigen identical with MPMV p27 (18, 81), proviral sequences of BaEV, PO-1-Lu, and MPMV in Old whereas a third laboratory reported the J-96 isolate to World monkeys, SMRV proviral sequences, even under low- possess an antigen similar to but not identical with the stringency hybridization conditions (13), have been identi MPMV p27 (106). Because the J-96 cells possess HeLa cell fied only in squirrel monkeys and not in other New World markers (75) and because all viral isolates from HeLa or monkeys that share habitats. The absence of SMRV proviral suspected HeLa cells have thus far been shown to be sequences in other New World monkeys may indicate a identical with MPMV, further studies on the J-96 isolate may high degree of divergence among these animals making show this virus to be identical with MPMV. detection of these sequences difficult. Alternatively, SMRV Immunological analysis of viral RT also provides a proviral sequences may have been acquired by squirrel method for classifying retroviruses into distinct groups. monkeys after their divergence from other New World Antiserum prepared against partially purified RT of MPMV monkeys. An interesting observation has been reported was found to neutralize the endogenous polymerase activity recently (13) demonstrating the presence of gene se of MPMV (105), X-381 (105), and AO (81) viruses. The quences partially homologous to SMRV in the cellular DNA antiserum did not inhibit the RT of AMV, FeLV, R-MuLV, F- of normal tissues of the New World carnivore, the skunk MuLV, SSV, and MMTV (81, 105). (Mephitis mephitis and Spi/ogale putorius). The final extent The antigenic properties of the langur monkey isolate, of hybridization of SMRV cDNA to skunk DNA was 8 to 10% PO-1-Lu, have been compared with those of MPMV after homologous RIA's with the use of purified MPMV proteins under relatively stringent hybridization conditions and 15 to 17% under lower stringency conditions, whereas 100% p27 and gp70 and antiserum to MPMV (99). PO-1-Lu con hybridization was achieved to squirrel monkey DNA under tained an antigen that was antigenically indistinguishable either conditions. No sequence homology was observed from MPMV p27. In contrast PO-1-Lu failed to compete in with other members of the family Mustelidae (mink and the assay for the MPMV envelope protein gp70. PO-1-Lu weasel), as well as other New World carnivores, including also has been reported not to exhibit cross-reactivity in RIA's for the major structural proteins of any of the known racoons and various cats (13). type B and type C retroviruses (99). In contrast to PO-1-Lu, the squirrel monkey isolates Immunology SMRV and M534 exhibited no antigenic cross-reactivity in homologous RIA's for both MPMV p27 and gp70 structural Evidence for the uniqueness of MPMV, in comparison to other members of the retrovirus genera, was originally proteins (41, 92, 99). Initial studies with both species- and derived from immunological studies of the virus in which interspecies-specific immunodiffusion assays revealed no immunodiffusion was used (5, 77). The development of antigenic relatedness between SMRV and MPMV, MMTV, sensitive RIA's for the virion structural proteins p27, p15, BaEV, SSV, R-MuLV, RD114, BLV, EIAV, FeLV, GALV (92), and B-GPV.4 More sensitive interspecies RIA's have been and gp70 has greatly expanded our understanding of the relatedness of MPMV to other retroviruses. Table 5 presents reported that do detect cross-reactivities between MPMV, a summary of results for the homologous assays for these 3 BaEV, RD114, SMRV, and PO-1-Lu. With the use of a broad

3132 CANCER RESEARCH VOL. 38

Table 5 Immunological cross-reactions " between MPMV and other retrovirus isolates

Homologous RIA for MPMV structural pro Interspecies RIA teins

and '"l-la- p27 and 125I- and I25l-la- '"l-labeled beled BaEV labeled beledSMRVgp70+NDNDNDNDND+NDND_NDND_+__NDNDND+ND_NDND_NDND VirusType p27p15+ gp70++ 9P70++ SMRVp35++ DMPMVX-381FTP-1AOJ96PO-1-LuSMRVM534Type 6++ ND+ c+ c+ c+ rND+ rNDND-_-ND_ND____NDNDND_NDNDNDNDNDNDNDAnti-MPMVrNDND++ rNDND++NDND_NDND____NDâ€”ND_NDâ€”_-â€”NDNDAnti-MPMV ND+ ND+ NDNDNDND_ f+ND__ND_+__NDNDND+NDNDNDNDNDNDNDAnti-MPMV

CF-MuLVR-MuLVAKR-MuLV"RaLVFeLVRD114SSVGALVPK-15AMVRAV-2BaEVEIAVBLVM432EWCVType

_â€” _- _- _- _â€” _- _NDNDNDNDNDNDND

NDND NDâ€”

BM MTVB-GPVType â€”¿ND-e

EVisna'"l-labeled ND'Â«l-labeled â€¢¿Compiledfrom Refs. 18, 28, 31, 32, 36, 54, 90, 92, 96, 101, and 106. * +, homology; -, no homology; ND, not done. c D. Fine, G. Schochetman, S. Devare, and J. Stephenson, unpublished data. d AKR-MuLV, AKR murine leukemia virus; RaLV, rat leukemia virus; PK-15, pig virus; EWCV, European wildcat virus. e Represents lack of cross-reactivity of MPMV p27 in homologous assay for Visna virus p27 (104).

interspecies RIA, with goat antiserum to disrupted MPMV that natural sera from both Old and New World monkeys and 125l-labeled BaEV gp70, a cross-reactivity was reported contain antibodies that recognize the retroviruses derived between MPMV and the 2 type C retroviruses, BaEV and from that species (e.g., SMRV and MPMV) and also exhibit RD114 (96). This reactivity did not extend to other type C interspecies cross-reactivities with MPMV, BaEV, SMRV, viruses, GaLV, SSV, FeLV, R-MuLV, and AKR murine leu and RD114. Table 6 summarizes the species and interspe kemia virus (96). Subsequent studies with this broad inter cies reactivities determined with natural sera from prosimi- species assay have demonstrated that PO-1-Lu and SMRV ans, Old World monkeys, New World monkeys, and apes. It also share cross-reactivity with the gp70's of MPMV, BaEV, is evident that in some but not all of the animals tested thus and RD114 (99). However, interspecies RIA's for SMRV p35 far, naturally occurring antibodies recognize specific deter and MPMV p27, with both goat and rabbit antisera to SMRV minants of the major nonglycoproteins shared by the type and MPMV, demonstrated cross-reactivity only between D retroviruses of both Old World (MPMV) and New World MPMV, SMRV, and PO-1-Lu (28, 31, 54). No cross-reactivity (SMRV) monkeys, but not with the p30 of BaEV. Initial was observed in these assays for the pSO's of mammalian indications that similar immunological reactivities also re type C (SSV, BaEV, RD114) or type B (MMTV, BLV) retrovi sided with the envelope proteins of these viruses, in addi ruses (28, 31, 54). Conversely, in interspecies assays for tion to BaEV, were suggested by the demonstration that pSO's of mammalian type C viruses, MPMV shares no natural sera cross-reacted with MPMV, SMRV, and BaEV detectable cross-reactivity (82). These findings are signifi with the use of intact virus competition RIA's (41). A subse cant, especially in view of the recent demonstration (31, 36) quent study (32) in which purified envelope glycoproteins

OCTOBER 1978 3133

Table 6 Species and interspecies cross-reactivities of primate retroviruses with sera from various primates SMRVProsimians MPMV

ofsera(Ga/ago virus0/13p27 p350/13BAEVWholeWhole virus virus p30

Bush babyOrigin crassi- caudatus)Whole

Old World monkeys Macaque (M. mulatta) 20/121" (40- 25/121 (20- 1/10(400) 5/121 (20-80) 0/75 20.250)'' 10,240) (Macaca fascicu- 6/21 (400- 1/2(2,560) 3/19 0/2 0/2 laris) 51,200) (Macaca spe 0/10 ciosa) Grivets (Cercopithecus 3/19 3/19 aethiops) Baboon (Pap/o cynoceph- 7/15(40- 0/10 7/15(40- 0/10 7/10(200-1,600) 0/10 alus) 20.250) 20.250) (Pap/o anubis) 1/11 1/11 (Pap/o hamad- 0/2 1/2 ryas) Mangabey (Cercocebus atys) 0/2

New World monkeys SquirrelCapuchinOwlMarmosetSpiderHowlerApesChimpanzeeGorillaOrangutan(S.sciureus)(Cebus 0/140/60/20/10(>40) 2/14(40-80)2,050)2/6 capuci-nus)(A. (250-750)0/23/9

trivirgatus)(Saguinus oedi-pus)(Saguinus 0/53/12 0/45/12 fusicol-lis)(Saguinus (100-200)1/3(100)0/60/60/719/122(250-(100-200)1/31/60/60/70/30/70/40/120/5

nigri-collis)(Callithrix

jac-chus)(Ã•teles

sp.)(A. belzebÃ¹!)(P.

troglodytes)(Gorilla gorilla)(Pongo pygma-cus)14/122

" Number of animals positive/number tested. 6 Numbers in parentheses, range of positive serum titers expressed as a reciprocal of serum dilution capable of binding >25% of the labeled antigen.

(gp70) of SMRV, MPMV, BaEV, RD114, and hyperimmune possible that these determinants represent highly con sera were used demonstrated that the interspecies reactivity served amino acid sequences that were present in a progen observed with intact virus resided at least in part with this itor virus common to all primate type D retroviruses even antigen. The findings with the natural sera are in agreement though they share no detectable nucleic acid sequence with those studies in which hyperimmune sera directed homology. This progenitor type D virus would have to have against their respective viruses were used. The prevalence been integrated within the genome of an ancestor common of antibodies cross-reactive with type D retroviruses is to both Old and New World monkeys prior to their diver evident from the fact that they have been found in newly gence approximately 50 million years ago (Chart 1). Fur imported rhesus monkeys as well as in rhesus monkeys thermore, this progenitor virus would have been distinct bred in captivity in geographically separated primate cen from the progenitor virus of the endogenous type C primate ters throughout the United States. retroviruses because of the lack of any cross-reactivity of the BaEV p30 with the analogous proteins of PO-1-Lu, Evolutionary Prospectus for Type D Viruses in Primates SMRV, or MPMV. Evidence for an earlier common progeni tor virus for both type C and type D retroviruses could be Based on the cross-reactivity observed between the major argued from the observations of interspecies reactivities of nonglycoproteins of the distantly related primate endoge the envelope glycoproteins of PO-1-Lu, SMRV, and now nous retroviruses of langur and squirrel monkeys (28), it is BaEV. Conservation of the 3-dimensional structures under-

3134 CANCER RESEARCH VOL. 38

Endogenous Exogenous Primate Group Subfamily Genus Virus Virus Immunological Assay Group Interspecies Interspecies p2T-p55 gpTÃŒT

I Alouatta New World ICebus I Aolus l Lagothrix Â»SaimirÃ - SMRV

Gorilla Pan Homo

Pongo Hylobates

Presbytls - Po-1-Lu Colobus Type D Virus Integration Erylhrocebus Cercopithecus

Cercocebus Macaca Mandrillus Papio BaEV 50 40 30 20 10 0 Millions of Years Chart 1. Evolution of type D retroviruses of Old and New World monkeys based on immunological cross-reactivities (see "Immunology"), -, cross- reactivities between endogenous viruses; . additional cross-reactivities between the endogenous viruses and the exogenous virus, MPMV. lying these reactions may have enabled the viruses to gain derived from the fact that MPMV was initially isolated from a selective advantage for survival either by protection from a spontaneous mammary tumor of a nonhuman primate. immunological surveillance by a host carrying an endoge These studies have attempted to provide evidence for the nous virus with similar sequences or by the ability of the presence of whole virus or viral components (proteins and/ virus to infect evolutionary descendents derived from the or nucleic acids) in human breast tumors. Although many host carrying the earliest common progenitor virus. Evi of the studies have indicated the presence of viral compo dence supporting the latter hypothesis may derive from the nents, none of the individual positive findings have been reported ability of RD114 (which was derived from BaEV) reproduced in other laboratories. The various reports of gp70 to block MPMV infectivity in KC cells, suggesting that positive findings of MPMV in fresh human material (as these viruses share common receptor sites (see "Infectiv compared to continuous cultured cell lines) can be sum ity"). This observation is important in light of the cross- marized as follows. reactivity between gp70's of RD114 and BaEV (96). There 1. Sequence homology was reported between MPMV fore, one might also predict that MPMV, PO-1-Lu, SMRV, [3H]cDNA and RNA from human malignant breast tu BaEV, and RD114 all share common determinants that are mors (27). In the same studies no sequence homology possibly involved in adsorption to specific target cells and was observed with the same probe and RNA of benign that are also the same determinants involved in the inter- breast tumors or normal tissues. However, sequence species cross-reactivity between their viral glycoproteins. homology was assayed by hydroxylapatite chromatog- However, we cannot rule out the possibility that the cross- raphy and cesium sulfate centrifugation. These assay reactivities of the envelope proteins were derived from a techniques could artificially yield high degrees of recombination of sequences coding for the gp70's of a type hybridization as compared to analysis by S, nuclease D (e.g., PO-1-Lu) and a type C (e.g., BaEV) retrovirus. This digestion. In fact, with the S, technique, no sequence event would confer the same selective advantages as those homology was observed between MPMV [3H]cDNA that might have arisen by the evolutionary conservation and RNA of human malignant breast tumors (102). process discussed above. The demonstration of these inter- Further studies with MPMV cDNA probes representing species cross-reactivities that are shared by distantly re the entire viral genome and in which alternate ap lated primate retroviruses, particularly those associated proaches are used to detect hybrid formation will be with major envelope proteins, provides a means for detect required to resolve these discrepancies. ing determinants that are representative of all primate 2. With the use of a sensitive RIA for MPMV p27, anti- retroviruses presently known and yet to be isolated. Thus, genie reactivity related to this protein was reported in these assays may provide useful tools for the detection if 8 of 18 partially purified preparations derived from not the isolation of a human retrovirus. human breast tumors (107). In the same study no reactivity was detected in normal placental tissues or Type D Viruses in Humans in tumors that were not of breast origin. In contrast a similar study (18) with analogous procedures reported The majority of the studies published on the presence of no MPMV p27 reactivity in 15 breast carcinomas ex type D viruses in humans have been concerned primarily amined. The second study used individual columns with MPMV and its role in human neoplasia, particularly for the purification of cross-reactive antigens from mammary neoplasia. The numerous attempts to implicate each tumor to eliminate any residual antigen carryover MPMV in the etiology of mammary neoplasia are obviously from virus purification. A protease inhibitor was also

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included in their RIA buffers to eliminate false-positive presented data extending our knowledge of MPMV. The reactions due to proteolytic activity. Negative findings first study (W. Drohan, D. Colcher, and J. Schlom. The Use were also reported with the less sensitive technique of of Standard and Relaxed Hybridization Conditions to Detect immunodiffusion (77). Two Classes of Sequences Related to Type D Retroviruses 3. Antibodies reactive with 125l-labeled MPMV p27 were in the DNA's of Primates. Biochim. Biophys. Acta, in press) detected in plasma from patients with various cancers, demonstrated the existence of two classes of MPMV-related including acute myelogenous leukemia and breast sequences in the DNA's of various Old World monkeys. One cancer, as well as from individuals without evidence class consists of the previously reported (34) sequences of cancer (63). These results were not substantiated in highly related to about 20% of the MPMV genome. The 2 independent studies (18, 31). In the latter 2 studies, sequences in the second class are related to an additional assays were performed on a combined total of 240 20 to 40% of the MPMV genome and are detected only sera collected from individuals with various cancers under low stringency conditions of nucleic acid hybridiza (including 54 with breast cancer), with other disorders, tion. These latter sequences represent highly diverged and with no diagnosed disease. MPMV sequences as evidenced by the low thermal stability 4. Ohno ef al. (79) reported the immunological cross- of the hybrid duplexes and provide evidence that numerous reactivity of RT from MPMV with a Mg2'-preferring type D viruses exist in the primate population. The second DMA polymerase previously purified from human study (D. Colcher, Y. A. Teramoto, and J. Schlom. Immuno breast cancer particles (80). Cross-reactivity was ob logical and Structural Relationships between Langur Virus served by both inhibition of enzyme activity and com and Other Primate Type D Retroviruses. Virology, in press) plex formation between the purified human particle demonstrates that although MPMV and PO-1-Lu cannot be enzyme and anti-MPMV RT IgG. No such interactions distinguished by homologous RIA's for their p27 structural were observed with RT's of AMV, R-MuLV, F-MuLV, protein, they can be readily distinguished in a RIA for their FeLV, SSV, and MMTV (79). In the same studies, DNA p10-p12 proteins. This provides an additional type-specific polymerase-containing particles isolated from spleens assay to distinguish between the two viruses. of chronic myelogenous leukemia, chronic lympho- cytic leukemia, and Hodgkin's disease showed no Acknowledgments cross-reactivity with anti-MPMV RT IgG. Although the We wish to thank our colleagues. Dr. R. Gilden, Dr. H. Rabin, Dr. L. association of this antigenic reactivity with breast Arthur, and Dr. R. Massey, for critical evaluation and suggestions and M. A. Gonda and M. Gregg for preparation of electron micrographs. We are tumors has potential applications as a diagnostic tool, grateful to M. Loose for considerable assistance in the technical preparation currently the assay is cumbersome and lacks sensitiv and editing of the manuscript. We also wish to acknowledge P. McLane, L. ity and reproducibility from specimen to specimen. Conners, and J. Clarke for help in typing the manuscript. 5. Particles resembling MPMV have been observed by References electron microscopy in negatively stained prepara tions of human milk concentrates (20, 88). In these 1. Abrell, J. W., Smith, R. G., Robert, M. S., and Gallo, R. C. DNA Polymerase from RNA Tumor Viruses and Human Cells: Inhibition by combined studies 841 milk samples collected from 723 Polyuridylic Acid. Science, 777: 1111-1113. 1972. women with and without familial history of breast or 2. Ahmed, M., Korol, W., Larson, D. L., Harwood, K. R., and Mayyasi, S. other types of cancer were examined for the presence A. Interactions between Endogenous Baboon Type C Virus and Onco- genic Viruses. Part 1. Syncytia Induction and Development of Infectivity of particles morphologically resembling MPMV. Al Assay. Intern J. Cancer, Ã•6:747-755, 1975. though MPMV-like particles were observed in 184 of 3. Ahmed, M., Korol, W., Schidlovsky, G., and Mayyasi, S. Defective Virus the samples, no differences were observed between Clones of Mason-Pfizer Virus. Proc. Am. Assoc. Cancer Res., 17: 165, 1976. the incidence of occurrence of these particles in milks 4. Ahmed. M., Korol, W., Yen, J., Schidlovsky, G., and Mayyasi, S. A. from women with familial history of breast or other Detection of Mason-Pfizer Monkey Virus Infection Using Human KG Cells Carrying Rous Sarcoma Virus Genome. J. Nati. Cancer Inst., 53: types of cancer and those from women without such a 383-387, 1974. history. At present the actual identity of such milk- 5. Ahmed, M., Mayyasi, S. A., Chopra, H. C., Zelljadt. I., and Jensen, E. associated MPMV-like particles is unclear since they M. Mason-Pfizer Monkey Virus Isolated from Spontaneous Mammary Carcinoma of a Female Monkey. Part 1. Dectection of Virus Antigens have no reported biological activity (e.g., infectivity by Immunodiffusion, Immunoflurorescent, and Virus Agglutination for cell cultures) and thus may represent cell frag Techniques. J. Nati. Cancer Inst., 46: 1325-1334, 1971. 6. Ahmed, M., Schidlovsky, G., Korol, W., Vidrine, G., and Cicmanic, J. L. ments. Occurrence of Mason-Pfizer Monkey Virus in Healthy Rhesus Monkeys. These conflicting studies on the association of MPMV Cancer Res., 34: 3504-3508, 1974. with human neoplasia indicate the need for further studies 7. Ahmed, M., Yen, J., Holden, H. E., Korol, W.. Schidlovsky, G., and with new and more sensitive techniques. The broad inter- Mayyasi, S. A. Characterization of Mason-Pfizer Monkey Virus Induced Transformation in Vitro. Arch. Virol.. 55. 93-105, 1977. species assays (28, 32, 36, 41, 54, 96, 99) initially developed 8. Andzhaparidze, O. G., Liozner, A. L., Korchak, O. B., Melenikova. N. to identify immunological cross-reactivities between the L., Lotte, V. D., and Stepanova, L. G. Immunological Characteristics of Oncornavirus Low Passage Level Produced by T-9 Human Cell Line. major glycoproteins of type D viruses and other distantly Vopr. Virusol., 6: 660-666, 1976. related primate retroviruses such as type C viruses may 9. August, J. T., Bolognesi, D. P., Fleissner, E., Gilden, R. V., and Nowinski, R. C. A Proposed Nomenclature for the Virion Proteins of provide the most sensitive test systems for detection of Oncogenic RNA Viruses. Virology, 60: 595-601, 1974. human retroviruses. 10. Bauer, H., Daams, J. H.. Watson, K. 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Fig. 1. Comparative morphology of type B, C, and D retroviruses. a, cluster of Â¡ntracytoplasmic type A parti cles (representing immature precursor of budding type B virus) with doughnut-shaped nucleoid and electron- lucent center; Ã¹,immature type B virus with doughnut- shaped nucleoid and electron-lucent center budding from cytoplasmic membrane; c, mature, extracellular type B virus with eccentric electron-dense nucleoid and characteristic surface spikes; d, lack of morphologically recognizable Â¡ntracytoplasmic form for type C virus, e, immature type C virus with crescent-shaped nucleoid budding from cytoplasmic membrane; f, mature, extra cellular type C virus with centrally located condensed nucleoid; g, intracytoplasmic type A particle with elec tron-dense, doughnut-shaped nucleoid and projections on the outer perimeter of the nucleoid (representing immature intracytoplasmic precursor form of type D virus); h, immature type D virus with doughnut-shaped nucleoid with electron-lucent center budding from cyto plasmic membrane; I, mature extracellular type D virus with condensed bar-shaped nucleoid. All micrographs * d are approximately x 87,000. Thin sections were double- stained with uranyl acetate and lead citrate. Type B viruses were MMTV from C3H/Cgrl mammary adenocarcinoma cell cultures. Type C viruses were Theilen strain of FeLV cultivated in FL74 feline lymphocytes. Type D viruses were MPMV grown in A204 cultures.

OCTOBER 1978 3139

Donald Fine and Gerald Schochetman

Cancer Res 1978;38:3123-3139.

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