type d primate retroviruses: a review1 - cancer...

[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 theMason-Pfizer monkey virus (MPMV). MPMV was originallyisolated from a breast carcinoma of a female rhesusmonkey (an Old World monkey). MPMV is of obviousimportance in that it is the only retrovirus thus far isolatedfrom a mammary tumor of a primate and has been shownto have transforming potential for primate cells in vitro.Subsequent to the isolation of MPMV, viruses morphologically and Â¡mmunologicallyindistinguishable from MPMVhave been isolated from normal placenta and lactatingmammary glands of other rhesus monkeys in captivity.Recently, viruses morphologically resembling MPMV havebeen isolated from a langur monkey (another Old Worldmonkey) and from squirrel monkeys (a New World monkey). Based on nucleic acid hybridization studies, thelatter 2 viruses represent endogenous viruses in theirspecies of origin, whereas MPMV appears to be a horizontally transmitted virus containing gene sequences partially related to the langur monkey isolate. Studies on theimmunological relatedness of the type D retroviruseshave demonstrated interspecies cross-reactivities between the major internal and external proteins of theviruses. Furthermore, these viruses also share cross-reactivity of their major external glycoproteins with thoseof the type C baboon endogenous virus. These interspecies reactivities can also be demonstrated in natural serafrom both imported and laboratory-bred monkeys. Thedemonstration of these interspecies cross-reactivitiesshared by distantly related primate retroviruses providesa means for detecting determinants that are representative of all primate retroviruses presently known and yet tobe isolated and may provide new assays for detection ofa human retrovirus.

techniques. The retrovirus associated with the induction ofmammary cancer in mice, MMTV,2 is morphologically dis

tinct from the type C viruses and is the prototype of the typeB virus group (30). A retrovirus group of primate origin,which has properties similar to the type C and type Bviruses but is morphologically distinct from them, has beendesignated genus Oncornavirus D (30). The prototype virusof this genus is the MPMV. MPMV was originally isolatedfrom a mammary carcinoma of a female rhesus monkey (anOld World monkey) (19, 60). Because mammary carcinomasrepresent the most prevalent form of neoplasia in humansand, furthermore, because the incidence of mammary tumors in subhuman primates is rare (61, 62, 70, 78), MPMVis of obvious potential importance because it is the onlyretrovirus thus far isolated from a primate mammary tumor.

Recently, there have been reports that viruses morphologically and immunologically similar to MPMV have beenisolated from certain rhesus monkey and human tissuesand cell lines (6, 8, 10, 44, 49, 55, 57-59, 72, 81, 107-109).Virus morphologically resembling MPMV have also beenisolated from normal tissues of a langur monkey (99) (another Old World monkey) and from squirrel monkeys (53,99) (a New World monkey). Only the langur isolate sharessignificant immunological cross-reactivity and nucleic acidsequence homology with MPMV. The discovery of these 2viruses makes it apparent that MPMV is a member of alarger group of viruses of both Old and New World primates. Furthermore, the large accumulation of informationon MPMV and the new type D primate isolates warrants acomprehensive review of these viruses. This review willconsider the unique characteristics of type D retrovirusesas well as their distribution in the primate population andtheir relevance to human cancer.

Introduction

Over the past 60 years, a considerable amount of information has been amassed concerning the role of viruses asetiological agents of animal neoplasia. In particular, thoseviruses known as RNA tumor viruses or retroviruses havebeen implicated in a variety of cancers in a wide variety ofanimal species. Those associated with the induction oflymphomas, leukemia, or sarcomas have been termed typeC viruses based on their morphology as determined byelectron microscopy (14). They have been isolated fromseveral vertebrate classes and are structurally, biochemically, and biologically similar, although they are distinguishable by sensitive immunological and biochemical

1 This work was supported by the Virus Cancer Program, Contract N01-CO-75380, National Cancer Institute, NIH, Bethesda, Md. 20014.

Received April 5, 1978; accepted June 16, 1978.

History

MPMV. MPMV was first observed and isolated from atissue biopsy performed on an 8-year-old female rhesus

2 The abbreviations used are: MMTV, mouse mammary tumor virus;MPMV, Mason-Pfizer monkey virus; CMMT, cocultivated monkey embryoand mammary tumor; PO-1-Lu, langur virus; SMRV, squirrel monkey retro-virus; RT, reverse transcriptase; A204, human rhabdomyosarcoma; B-GPV,guinea pig endogenous virus; BaEV, baboon endogenous virus; AMV, avianmyeloblastosis virus; BLV, bovine leukemia virus; EIAV, equine infectiousanemia virus; oligo(dT)-poly(rA), noncovalent copolymer of oligodeoxythy-midylate and polyriboadenylate; oligo(dG)-poly(rC), noncovalent copolymerof oligodeoxyguanylate and polyribocytidylate; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; gp, p, followed by 2 numbers,glycoprotein and protein, respectively, followed by 2 numbers designatingmolecular weight in thousands (e.g., gp70, p27); gp69/71, glycoprotein witha molecular weight range of 69.000 to 71,000; RD114, feline endogenousvirus; KC, human glioma; RHFS, rhesis monkey foreskin; RIA, radioimmuno-assay; FeLV, feline leukemia virus; cDNA, complementary DNA, SSV, woollymonkey virus; GALV, gibbon ape virus; BaLV, baboon leukemia virus;F-MuLV, Friend leukemia virus; R-MuLV, Rauscher leukemia virus.

OCTOBER 1978 3123

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D. Fine and G. Schochetman

monkey (Macaca mulatta). This animal had developed aspontaneous tumor (69) in the region of the left mammarygland after being in a state of continuous estrus for almost1 year. The animal was sacrificed approximately 2 monthsafter the discovery of the tumor, at which time virus wasobserved in tumor samples by thin-section electron microscopy (19, 23). At necropsy the tumor was found to havemetastasized into both breasts, the left axilla, flank, and ribcage. Secondary tumors were also found that involved anumber of organs including an ovary, the adrenals, pancreas, kidney, liver, stomach, and lymph glands. The primary tumor histologically resembled a medullary carcinomalacking lymphoid stroma (69). Because the tumor did notclosely resemble conventional breast adenocarcinomas microscopically and because surrounding breast parenchymadid not present any evidence of developing in situ malignantchanges, the possibility was raised that the tumor arose asan intramammary anaplastic lesion (M. Black, personalcommunication).

Since the tumor specimen grew poorly when placed intoculture, virus isolation was effected by cocultivation ofminced tumor tissues with primary or early-passage monkeyembryo cell cultures (60). This led to the establishment of acontinuous cell culture system (CMMT) which served as aproducer of MPMV. Cultures of CMMT cells transferred formore than 95 passages consist of cells with distinct rhesusmonkey karyotype (74). Although CMMT cultures wereoriginally maintained by adding nonirradiated rhesus embryo cells to the MPMV-infected cells every 30 days, highpassages of the culture grew without further cocultivation(74). Virus from the original tumor was successfully transmitted as a cell-free filtrate with the use of low-passagemonkey embryo cells (60).

To determine whether MPMV or MPMV-related viruseswere present in tissues of apparently normal rhesus monkeys, Ahmed et al. (6) examined lactating breast tissue,placentas, and fetuses of monkeys in a closed breedingcolony at Kensington, Md. These workers were successfulin isolating 2 new MPMV-related isolates; one, designatedX-381, was derived from a lactating mammary gland biopsy,and the other, designated FTP-1, was derived from placen-tal tissue cocultivated with human NC37 lymphoblastoidcells. Although MPMV antigens and budding MPMV-likeparticles were observed in 3 other mammary biopsy cultures, they were lost as a result of simian foamy viruscontamination (6).

The fact that viruses similar to but not identical withMPMV could be isolated from normal rhesus monkey tissueraises the possibility that in the original monkey tumortissue MPMV was present fortuitously and was not etiologi-cally involved in tumor development. Consistent with thispossibility was the fact that virus particles morphologicallysimilar to MPMV were also observed in several nontumor-ous tissues, including the thymus and the lymph nodes ofthe original tumor-bearing animal (23).

A virus thus far indistinguishable from MPMV was alsoisolated from HeLa cell cultures by Bauer ef al. (10, 44,103). The presence of MPMV in these HeLa cell culturesprobably represents a laboratory contamination subsequentto the establishment of the cell line. This conclusion isbased on the fact that MPMV provi ral sequences are not

present in all HeLa cell stocks, as measured by nucleic acidhybridization, but are present in those cultures expressingMPMV (25). Numerous isolations of MPMV-like viruses froma variety of human cell cultures have been reported; Table1 lists the names and sources of the various isolates.Recently, many of these cell lines were shown to possessHeLa cell chromosomal and isoenzyme markers (65, 75). Itis possible that these cell cultures are examples of cross-contamination of the original cell cultures with HeLa cells,and it seems reasonable to assume that the virus isolatesalso represent laboratory contaminants.3 Interestingly,

these virus isolations occurred in separate laboratoriesaround the world and, supposedly, none of the researchgroups was conducting research on MPMV. These strikingobservations indicate the widespread distribution of virusesindistinguishable from MPMV and demonstrate the propensity of these viruses for infection of human cells. Thesefeatures dictate the need for extreme caution in studyingprimate-derived retroviruses with regard to their role in theetiology of human cancers.

PO-1-Lu. A new retrovirus resembling MPMV was isolatedfrom another Old World monkey, Presbytis obscurus, thespectacled langur (99). The virus, designated PO-1-Lu, wasrecovered from cultures of lung tissue cocultivated with batlung cells (TbILu) and could be distinguished from MPMVbased on differences in host range, antigenic characteristics, and nucleic acid sequence homology. Complete pro-viral sequences of PO-1-Lu have been found in langurmonkeys, indicating that the virus is endogenous in thisspecies (12).

SMRV. Another retrovirus, SMRV, was isolated from fetallung tissue of SaimirÃsciureus, a New World monkey (53).The isolation was achieved by cocultivation of fetal lungtissue with canine thymus (Fcf2th) cells. Additional virusesresembling SMRV were isolated from a variety of tissues ofS. sciureus by the cocultivation technique (53, 99). Theoriginal isolates derived from squirrel monkeys have beendesignated SMRV (53). A subsequent squirrel monkey virusisolate has been referred to as M534 (99). Although thevirus contains a Mg2+-preferring RT and is morphologicallysimilar to MPMV, it shares neither group-specific immuno-logical relatedness nor nucleic acid sequence homologywith MPMV. The ability to isolate SMRV from a variety oforgans of more than 1 squirrel monkey (53) and the factthat all tissues of squirrel monkeys contain SMRV proviralsequences (26, 41, 54, 91) demonstrate that SMRV is anendogenous virus of this animal species. A recent report(100), which indicates that the isolation of endogenousprimate viruses from species other than baboons is difficult,stresses the importance of the isolation of both SMRV, thefirst endogenous virus from a New World primate, and PO-1-Lu, the first non-type C endogenous virus from an OldWorld primate.

Virus Classification

In 1974, Retroviridae was established (30) as a family ofviruses that contain a 60 to 70S single-stranded RNA and an

3 For proper referencing we have referred to each virus isolate accordingto the cell designation used in the original isolation although, as stated,many if not all of the cell cultures are of HeLa cell origin.

3124 CANCER RESEARCH VOL. 38



Type D Primate Retroviruses: A Review

Table 1Type D primate retrovirus isolates

OriginSubhuman

primatesM.mulattaP.

obscurisS.

sciureusHuman

(Homo sapiens)Virus

descriptionMPMVX-381FTP-1PO-1-LuSMRVM534J96HEp-2He

LaHeLa-ACaOvAODAPTRHDetroit

6GraffiHost

tissuedescriptionSpontaneous

mammarytumorofa female rhesusmonkeyLactating

mammary gland ofafemalerhesusmonkeyPlacentaof a femalerhesusmonkeyLung

tissue of a spectacledlan-gurLung

tissue of a squirrelmonkeyLungcells of a squirrelmonkeyLeukemiaEpidermoid

carcinomaCervicalcarcinomaClone

of cervicalcarcinomaOvariancarcinomaAmnionAstrocytomaSpontaneously

transformedrenalembryocellsBone

marrow of patientwithlungcarcinomaEmbryonic

cells from fetus derived from female with generalized portio carcinomaRef.606699539910957,

1091010958,1088110910915,59,7249

antigenically specific RNA-dependent DMA polymerase.termed RT. The various members of the family Retroviridaeare subdivided into 6 genera designated A through F, aclassification based largely on morphological distinctionsset forth by Bernhard (14). Type A particles, which are 60 to90 nm in diameter, consist of 2 forms, intracytoplasmic andintracisternal. Both types are double-shelled and have anelectron-lucent center. The role of the intracisternal Aparticle (which constitutes genus Cisternavirus A) is unknown; however, the intracytoplasmic A particles are considered to be precursors to the type B particles (30).Intracytoplasmic A particles (Fig. 1a) and type B particles(Fig. 1, b and c) are 2 morphological forms associated withmembers of the genus Oncornavirus B, generally termedthe type B virus. Type B particles bud at the cell membrane(Fig. 1b) with a complete nucleoid to form extracellularparticles, the mature form of which has an eccentric nucleoid and prominent surface spikes on its outer envelope(Fig. 1c). The members of the genus Oncornavirus C, morecommonly termed type C viruses, include those viruses ofvarious animal species that have no intracytoplasmic morphologically identifiable form (Fig. id) but that bud with acrescent-shaped nucleoid at the cell membrane (Fig. 1e).The extracellular form of the virus contains a central nucleoid and lacks surface projections or spikes (Fig. 1f ). Intheir description of the virus particles found in the rhesusbreast tumor from which MPMV was first isolated, Chopraet al. (19, 21, 23) observed both intracellular (Fig. 1, g andh) and extracellular (Fig. 1;) particle types. The intracellularparticles were ring shaped, measured 60 to 95 nm indiameter, and appeared near the plasma membrane. Theextracellular or "mature" particles, measuring 100 to 120nm in diameter, contained an electron-dense nucleoid andwere free as well as attached to membrane in intracellular

spaces. The latter particle contained both an outer unitmembrane and a distinct membrane binding the nucleoid.MPMV, which was subsequently propagated in humanlymphoblastoid (NC37) (60), A204 (48, 81), or rhesus monkey (MA-101, CMMT) (60, 64, 67) cell cultures, has exhibitedparticle types with these basic morphologies.

Prior to the establishment of the family Retroviridae,MPMV was considered to be morphologically similar toMMTV, the prototype type B virus (23, 64, 67). Chopraand Mason (23) first correlated MPMV with MMTV based ontheir observations that extracellular virions consistedlargely of enveloped particles containing an eccentricdense nucleoid and that virus development occurred intra-cytoplasmically in the form of "A" particles (Fig. 1g),

thought to be the precursor of the type B particle. AlthoughChopra and Mason (23) pointed out that the extracellularform of MPMV resembles the type C viruses (i.e., characteristically devoid of surface spikes), Kramarsky ef al. (64)found MPMV to be in the size range of MMTV (125 to 130nm), larger than the typical avian and murine type C viruses(110 nm) previously examined. The latter investigators alsodistinguished MPMV from MMTV on the basis of the size ofthe intracytoplasmic "A" particles (diameters of 90 and 75

nm, respectively) and the presence of knobs on the virionenvelope of MPMV, in contrast to the longer spikes on theenvelope of MMTV. On the basis of these distinguishingmorphological characteristics, MPMV was placed in thegenus Oncornavirus D (Fig. 1/). MPMV was also distinguished from the foamy viruses of monkeys and chimpanzees (22), which are grouped in the genus Spumavirus F, inthat the latter contain an electron-lucent nucleoid andprominent surface spikes ranging from 5 to 10 nm in length.

The rhesus monkey isolates X-381 and FTP-1 (6) and thelangur isolate PO-1-Lu (99) exhibit virus maturation identi-

OCTOBER 1978 3125




cal with that of MPMV. The virus particles observed incertain HeLa cell cultures (44) and in a cell culture derivedfrom human brain (55) were also indistinguishable fromMPMV. Virus particles morphologically resembling MPMVhave been observed in other cell cultures derived fromhuman tumors (49, 72, 81).

Although SMRV was originally reported to resembleMPMV morphologically (53), subsequent comparative electron microscopy studies (48) emphasizing thin-section andcritical-point drying techniques showed that the extracellular mature forms of the 2 viruses are distinguishable. SMRVis generally round with a central electron-dense nucleoid,whereas MPMV has an irregularly shaped envelope enclosing a tubular or barrel-shaped nucleoid.

Additional properties used to characterize retrovirusesinclude buoyant density analysis in different media anddivalent cation preference for the viral RT. MPMV (93), aswell as X-381, FTP-1,4 and SMRV (92), exhibit a buoyant

density of 1.17 g/ml in sucrose and 1.21 g/ml in CsCI. Thisproperty is also shared by the type B viruses, MMTV (89)and B-GPV (71), and at least 1 member of the primate typeC viruses, BaEV.4 In contrast the murine type C viruses

exhibit a single buoyant density of 1.16 g/ml in bothsucrose and CsCI (89).

MPMV (1, 33, 105), X-381 (105), FTP-1, SMRV (26, 54, 92),PO-1-Lu (99), and retroviruses isolated from HeLa (15, 103),HEp-2 (15), and AO (15) cells exhibit a strong preference forMg2* as the divalent cation for viral RT activity, whereas all

members of the murine and primate type C viruses havebeen shown to have a cation preference for Mn2+ (33, 95).Other viruses having RT's with a preference for Mg2*

include those of mouse, MMTV (33); avian, AMV (50);bovine, BLV (46); and equine, EIAV (17) origin.

Although various members of Oncornavirus B and Cpossess 1 or more of the above morphological, biophysical,and biochemical properties, only MPMV, SMRV, PO-1-Lu,X-381, FTP-1, and the HeLa isolate possess all of thefollowing properties: (a) budding from the cell membranewith a complete nucleoid; (b) intracytoplasmic A particles;(c) extracellular particles having a central nucleoid andlacking prominent surface projections; (d) buoyant densityof 1.16 g/ml in sucrose and 1.21 g/ml in CsCI; and (e) Mg2*-

preferring viral RT. The above properties are summarized inTable 2 for representative retroviruses. On the basis of thisinformation, we suggest that those viruses that possess allof the properties in common with MPMV be placed in thegenus Oncornavirus D.

Virus Structure

Like other retroviruses, MPMV contains a 60 to 70S RNA(52, 67, 94), which has a molecular weight of approximately8 x 106daltons when analyzed by electrophoresis in poly-acrylamide-agarose gels (94). MPMV 60 to 70S RNA's fromshort-term (2-hr) harvests, when denatured by heat (80Â°,2.5

min) or formamide (40%), yielded subunit structures of 2.8x 106daltons (94). Treatment of viral 70S RNA with limitingamounts of formamide resulted in a stepwise dissociationto intermediate RNA structures, suggesting that the MPMV

' G. Schochetman, unpublished data.

RNA subunits may be linked by differentially stable hydrogen-bonded RNA regions (94). Complete heat dissociationof MPMV 70S RNA also yielded 3 low-molecular-weightRNA species of 2.5 x 104, 3.5 x 104, and 1 x 10s daltons,corresponding to approximate sedimentation coefficientsof 4, 4.5, and 7S, respectively (94). These small RNA's are

similar to those found in other retroviruses.As reported for other retroviruses, MPMV contains a

polyadenylic acid sequence of approximately 200 nucleo-tides covalently linked to the viral RNA (47). Although notreported these sequences presumably are located at the 3'

end of the molecule.SMRV (26, 41, 91) and the MPMV-like isolates from AO

(16, 81), J96 (81), HEp-2 (109), and Detroit-6 (16, 72) cellsalso contain 60 to 70S RNA. However, relatively little hasbeen reported about the RNA structure of these and theother MPMV isolates.

Viral RT. The RT of retrovirusesconverts the viral geneticinformation, which is in the form of RNA, into a DNAmolecule which is subsequently integrated into the hostcell DNA and allows for the vertical transmission of the viralinformation in an unexpressed form as an integral part ofthe cell genome.

MPMV has been shown to have a virion-associated RT(1). The enzyme has been purified by a variety of techniquesincluding ion-exchange chromatography followed by gelfiltration (1, 56) and ion-exchange chromatography followed by affinity chromatography on polyribocytidylic acid-agarose (29). The molecular weight of the MPMV RT hasbeen reported to be 110,000 (1). A more recent estimate(29) placed the molecular weight at about 80,000, intermediate in size to the mammalian type C (m.w. 70,000) andtype B (m.w. 100,000) viral RT's (1, 56, 73). This latter

estimate is in good agreement with the molecular weightdeterminations for partially purified RT's from the SMRV(m.w. 80,000) (26) and PO-1-Lu (m.w. 85,000 to 90,000) (99).Both MPMV virion-associated and purified RT have beenshown to prefer Mg24 as the divalent cation in an endoge

nous reaction in addition to reactions utilizing the synthetictemplates oligo(dT)-poly(rA) and oligo(dG)-poly(rC) (29).

With the synthetic template oligo(dG)-poly(rC) oroligo(dT)-poly(rA), PO-1-Lu (99), SMRV (26, 53, 92), thevirus isolates from HeLa (103), and HEp-2 (58) cells werealso shown to prefer Mg2" as the divalent cation. The HeLa

cell isolate has also been reported to possess RNase Hactivity (103).

Structural Proteins. MPMV has been shown to contain 6major proteins with molecular weights of 68,000 to 70,000;27,000; 20,000; 14,000; 12,000; and 10,000 as determinedby SDS-PAGE (93, 101), similar to those reported for type Cviruses (9). On the basis of glucosamine labeling, the 68,000to 70,000 (gp70)-dalton protein is the major virion glycopro-tein and the 20,000 (gp20)-dalton protein is a minor glyco-protein (93). This is demonstrated by the fact that gp70 islabeled approximately 15 times better than is gp20 on amolar basis (93). Surface iodination of intact MPMV catalyzed by the enzyme lactoperoxidase demonstrated thatgp70 was the major external virion protein (93). gp20 hasbeen reported to have a molecular weight of 15,000 to17,000 by gel filtration in 6 M guanidine hydrochloride (93,101). This difference in molecular weight is probably due to





Table 2Properties of representative members of genera Oncornavirus B, C, and D

NucleoidType

B(MMTV)

Type C(MuLV)

Type D(MPMV)plasmic

Aparticles Atbudding+

Complete

Incomplete

+ CompleteIn

extracellularvirusEccentric,

denseCentricCentric,

denseSurface

projectionsLong

(spikes)ShortShort

(knobs)Buoyant

density(g/ml)Sucrose1.171.16

1.17Cesium

chloride1.21

1.16

1.21RT

cationpreferenceMg2+

Mn2+

Mg2+

the anomalous migration of glycoproteins in SDS-PAGE.That these 6 proteins are virus-coded and not host cell-derived components is indicated by observations that noneof the proteins was selectively lost during extensive purification of the virus and that all were present in the samerelative proportions, regardless of the cell type from whichthe virus originated (93). In addition p27 (the major nongly-coprotein) was found only in MPMV-infected cells and wasantigenically indistinguishable from virus grown in 3 different primate cell cultures (90, 93). These results furtherindicate that p27 is induced after virus infection and thusrepresents a virus-coded protein. Two additional proteins,gp48 and gp36, of uncertain origin, were found only in virusgrown in NC37 pells (93). Numerous type C viruses propagated in NC37 cells contain proteins of similar molecularweight (M. Ahmed, personal communication), which mayindicate that these proteins are host derived. However,additional studies are necessary to verify their origin. Thepossibility exists that 1 or both of these proteins representa differentially glycosylated form of the major glycoproteingp68. An example of this phenomenon has been reportedfor murine type C viruses, in which an incompletely glycosylated form of the major envelope glycoprotein gp69/71exists as a molecule with an approximate molecular weightof 45,000(gp45)(68).

The polypeptides of the 2 MPMV-related rhesus monkeyisolates X-381 and FTP-1 have also been examined andshown to be identical with those of MPMV. The FTP-1isolate, propagated in NC37 cells, also exhibited 2 additional glycoproteins, gp48 and gp36, as was observed forMPMV grown in NC37 cells (93). The HeLa isolate containedseveral low-molecular-weight proteins with molecularweights between 12,000 and 28,000. Also present were 2glycoproteins with molecular weights of 60,000 and 80,000(10). Although several of these proteins correspond to themajor proteins of MPMV, a more detailed characterizationof this virus is required to clarify this point.

The protein composition of MPMV and related isolates isclearly different from that of BaEV, a type C virus of OldWorld monkeys (97). At present the structural proteins ofthe PO-1-Lu isolate have not been examined. It would be ofconsiderable interest to determine whether PO-1-Lu, whichis morphologically indistinguishable from but only partiallyrelated antigenically to MPMV, contains structural proteinseither similar to or distinct from MPMV and BaEV.

The polypeptide composition of SMRV, the endogenousvirus of squirrel monkeys, was clearly distinct from that ofMPMV and MMTV (the prototype type B virus) as deter

mined by SDS-PAGE. Four major polypeptides with molecular weights of 40,000, 20,000, 14,000, and 8,000 wereresolved in virus propagated in human, mink, and caninecells (92). This result indicates that these 4 proteins arevirus coded and that the 40,000-dalton component is themajor nonglycosylated protein of SMRV. A subsequentstudy with agarose gel filtration under denaturing conditions (31) estimated the molecular weight of this protein tobe approximately 35,000; thus, this protein will be referredto as p35. SMRV propagated in mink cells also contained a73,000-dalton protein, presumably similar to the gp73 component of SMRV grown in A204 cells. In contrast SMRVfrom canine cells contained a 100,000-dalton polypeptideinstead of the 73,000-dalton component (92).

Biological Properties

Infectivity. Cocultivation of cells in culture has providedan important tool for the isolation of retroviruses, particularly of the type D family (6, 53, 60, 100). Although MPMVwas first isolated by cocultivation, it was also obtained bydirect infection of rhesus monkey embryo cells with tissueculture fluids from the original rhesus monkey breast tumorcell culture (60). This was the-first evidence that MPMV wasinfectious for primate cells and could replicate in vitro.Subsequent studies (Table 3) established that the hostrange of MPMV is primarily restricted to primate cell cultures, including those of human origin, although productiveinfections have been accomplished recently in cell culturesof horse epidermis (CCL57) (D. Fine, unpublished data) andAleutian mink lung (CCL64) (28). However, the horse epidermis cells produce approximately 10-fold less virus thando infected human cell cultures.

PO-1-Lu, like MPMV, also exhibits a restricted host range(Table 3) and replicates best in human (A204) and bat(TbILu) cell cultures (99). In contrast, SMRV has a broadhost range (Table 3) and can successfully replicate inhuman, rhesus monkey, and a variety of nonprimate cells(53, 92, 99). PO-1-Lu and SMRV share the common propertyof not replicating in cells of their respective host of origin(53, 99) and thus may represent examples of xenotropicprimate retroviruses.

Although infection of permissive cell lines with MPMVgenerally has no accompanying cytopathology (11, 60, 76),infection of rhesus monkey and human foreskin cultures(38, 40) and rhesus monkey embryonic lung cultures (Ref.38; E. Hunter, personal communication) resulted in theformation of multinucleated foci or syncytia in the cell

OCTOBER 1978 3127



Table 3Hosf range for type D retroviruses

VirusCell line designation and tissue

Species origin MPMV PO-1-Lu SMRV

PrimateOld World monkeys

Rhesus (M. mulatta)

African green (Cer-copithecus ae-thiops sabaeus)

Baboon (Papio cy-nocephalus)

New World monkeysSquirrel (S. sci-

ureus)Marmoset (Sa-

guinus sp.)Owl (/lofes trivirga-

tus)

Howler (Alouattabelzebul)

ApesChimpanzee (Pan

troglodytes)

Human (H. sapiens)

NonprimateHorse (Equus cabal-

lus)

ND" (mixed embryo)MA-101 (embryonic lung)Rhmk (embryonic kidney)RHFS (embryonic foreskin)DBS-fRhL-1 (lung)Vero (kidney)

LE-10 (leukocytes)

BFS (fetal skin)

SqMLu (lung)

MoLv (lung)MAK (kidney)OMK (kidney)

HMS (skin)

ND(lung)

SFRE:CL-1 (lung)LE-11 (leukocytes)A204 (rhabdomyosarcoma)RD (osteosarcoma)HOS (osteosarcoma)KC (glioma)HeLa (cervical carcinoma)AY603 (mammary adenocar-

cinoma)MCF-7 (mammary adeno-

carcinoma)NC37 (lymphoblast)HBL-100 (mammary epithe

lium)HE-37 (mixed embryo)HE-40 (mixed embryo)HSO410 (fetal fibroblast)HuFS (foreskin)HEK (embryonic kidney)WI38 (diploid)IMR-90 (diploid)

E. derm (dermis)

+ (60)+ (60, 67)+ "(7)

+ (3, 7, 38)+ (99)+ (60)

-(60)

+ (60, 67)

+ (60)-(60)+ (7, 11,81,99)+

+ (53)(99) + (99)

-(53)

-(53)

-(53)-(53)

-(53)

+ (53)

(99) + (53, 92, 99)

-J4.86)

-^(7,24,60)

+ (60)+ (60)

+ (38)+ (4, 7, 22, 48)+ (7, 86)

+ (53)

Mink (Mustelavison)Dog

(Canisfamili-aries)Cat

(Felisdomesticus)Bat(Tonatiabrasilien-sis)Hamster

(Mesocrice-tusauratus)Rabbit(Oryctolaguscuniculus)Mouse

(M.musculus)Rat(Rattusnorvegi-cus)MvlLu

(lung)MvlLu(64J1)(lung)Fcf2th(thymus)MDCK

(kidney)FEC(embryo)TbILu(lung)BHK

(kidney)SIRC

(cornea)LLC-RK,

(kidney)ND(lung)JLS-V9

(bonemarrow)NRK(kidney)+

(7,28)-(99)-(99)-(99)-

(7,99)-

(7,60)-(99)-(7)-(7)-

(7,60)-(7)-(99)-(99)-(99)+

(99)-(99)+

(53,92)+(99)+

(53, 92,99)+

(53)-(99)+

(99)+

(99)

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

3128




monolayer. Syncytia with similar morphologies are inducedby human and feline syncytia-forming viruses (51, 66) andRD114 (85). These syncytia were clearly distinguishablefrom those caused by simian foamy viruses by size (numberof nuclei per syncytium), time of appearance, and vacuoli-zation of the culture monolayer (38). Development of syncytia also occurs when KC cells (a Rous sarcoma virus-transformed human glioma cell line) are infected withMPMV, BaEV, and RD114 (2, 4, 85, 86). In RHFS and KCcells, the number of syncytia formed correlates with virusdose and provides the basis for a virus infectivity assay (4,38, 86). Syncytia formation occurs within 24 hr after infection in both cell lines (38, 86), is neutralized by MPMVantiserum (40), and is enhanced by Polybrene or DEAE-dextran treatment prior to infection. Syncytia persist in theMPMV-infected cultures for 2 to 4 weeks and then disappear, resulting in chronically infected cultures that areresistant to superinfection with MPMV (40, 86). SMRV wasinitially reported to induce syncytia on KC cells (53). However, this phenomenon is not reproducible, although thecopresence of SMRV with MPMV in the KC cells enhancesMPMV-induced syncytia (D. Fine, unpublished data). Asyet, PO-1-Lu has not been reported to induce syncytia in KCcells.

A recent study (E. Hunter, personal communication) demonstrated that UV irradiation of MPMV inhibited viral-induced syncytia in rhesus monkey embryonic lung cells andthat the inactivation followed single-hit kinetics. This resultexcludes a direct effect on the gp70 of the virus. Theinactivation of gp70 would be expected to follow multiple-hit kinetics. However, preexposure of KC cells to the gp70of RD114 blocked syncytia formation by both MPMV andRD114 (84). If the viral genome were the UV irradiationtarget, it would suggest that a functional viral genome wasnecessary for syncytia formation and would explain therelatively long time (approximately 24 hr) required forMPMV-induced syncytia formation. Therefore, the inhibition of syncytia formation by preexposure of KC cells to thegp70of RD114 may have resulted from saturation of specificreceptor sites shared by MPMV and RD114, thus preventingattachment and penetration of the virus. This is importantin light of the recent demonstration of interspecies cross-reactivity between the gp70's of MPMV and RD114 (96).

MPMV infections occur in the absence of syncytia formation in cell lines such as A204 (11), human osteosarcoma(Table 3), and SV40-transformed WI-38 (86). In A204 cultures, which are highly permissive for MPMV, newly synthesized virus is detected within 4 days after infection at inputmultiplicities significantly greater than 1 virus particle/cell.At lower input multiplicities (less than 1 virus particle/cell),MPMV infections occur that are inapparent until the cultures have been serially passaged several times. Productively infected A204 cultures carried over 30 passages afterinfection continue to express infectious virus into the extracellular fluid. MPMV production in the same infected cultures remains constant based on RT, MPMV p27, and virusparticle count determinations (11).

In Vitro Transformation. MPMV has been shown to possess in vitro transforming capacity for rhesus monkey,human, and horse cell cultures (3, 7, 40, 83). One characteristic of these transformed cells is their loss of anchorage

dependence, which enables them to grow in semisolidagar. In addition MPMV-transformed RHFS and horse epidermis cultures exhibit an altered morphology, loss ofcontact inhibition of movement, and chromosomal abnormalities (3, 7,40,83).

MPMV-transformed RHFS and horse epidermis cellschronically produced virus that was indistinguishable fromthe virus used in the original inoculum (CMMT-MPMV). Thisconclusion was based on the SDS-PAGE pattern of viralproteins (93) and complete identity of viral p27's (90) andgp70's (G. Schochetman, D. Fine, S. Devare, and J. Ste-phenson, unpublished data) in homologous MPMV RIA's.

The ability of MPMV-transformed cells to grow in semi-solid agar offers a means for determining frequency oftransformation and for isolation of transformed cell clones.Transformation in MPMV-infected RHFS cell cultures occurred at a low frequency (approximately 10 5) and wasdependent on multiplicity of input virus (3, 7, 83). Unin-fected RHFS cultures were fibroblastic and exhibited contact inhibition of movement, whereas sublines establishedfrom colonies of MPMV-transformed RHFS cells had apolygonal epithelioid phenotype, displayed loss of contactinhibition of movement, and contained foci of clumpedcells and syncytia. These transformed sublines grew 20 to90 times better in serum factor-free medium than diduninfected rhesus foreskin cells (83).

MPMV-transformed RHFS cells with epithelial morphology had a high plating efficiency in soft agar and exhibitedchromosomal abnormalities characterized by polyploidyand structural rearrangements, including chromosomalbreaks (7, 40). A second cell type, isolated from soft-agarcolonies of MPMV-infected RHFS cells in the studies ofAhmed ef al. (3, 7), had a fibroblastic morphology similar tothat of uninfected RHFS cells. The cultures showed lack ofcontact inhibition, a normal rhesus chromosomal complement, and a lower plating efficiency in soft agar. Bothepithelioid and fibroblastic transformed cell types producedintracellular MPMV antigens and virus particles, but thefibroblastic sublines generally produced >2 log]0 50% tissue culture-infectious doses/0.5 ml less infectious virusthan did the epithelioid sublines.

One fibroblastic subline of MPMV-transformed RHFScells was found to express MPMV antigen and virus particles that contained p27, but the virus recovered from theculture fluid lacked syncytia-forming capacity in KC cells(7). Negative-stain electron microscopy showed the morphology of the virus particles from this subline to bedistinguishable from KC-positive MPMV produced by othertransformed sublines of RHFS cells (7). KC-positive MPMVpossessed tails and condensed nucleoids, whereas KC-negative MPMV lacked tails and contained a doughnut-shaped nucleoid.

In Vivo Studies. Attempts to induce tumors by inoculationof MPMV into rhesus monkeys (37, 39), cynomolgus monkeys, squirrel monkeys, and marmosets have been unsuccessful (L. Wolfe, personal communication). This failuremay be a result of 1 or more of the following: (a) a requirement for a long latent period not yet reached in theinoculated animals; (b) the original virus inocula containedlittle or no transforming virus; (c) MPMV itself is notoncogenic but requires a helper virus or cocarcinogen; (d)

OCTOBER 1978 3129




MPMV is not oncogenic at all; or (e) effective immunesurveillance by the inoculated host. Consistent with the lastpossibility is the ability to detect high titers of both neutralizing and precipitating antibodies against the whole virusand several viral structural proteins in inoculated rhesusand cynomolgus monkeys (31). Despite the inability ofMPMV to induce tumors, many of the inoculated rhesusmonkeys became persistently infected, and virus could bereadily recovered from peripheral lymph nodes (37), saliva,and exfoliated cells in milk (A. Bogden, personal communication). Maternal transmission of virus via milk in rhesusmonkeys has been observed, providing evidence for at least1 mode of horizontal transmission.

Although rhesus monkeys neonatally inoculated withMPMV or MPMV-infected cells did not develop tumors,many developed severe lymphadenopathy, weight loss, andthymic atrophy (39). The consistent observation of thymicinvolution in the inoculated animals during the first severalweeks of age suggests that the thymus may serve as atarget organ for MPMV infection in infant monkeys. Theenhanced regression of the thymus may partially explainwhy a large proportion of the inoculated animals succumbed to secondary viral or bacterial pneumonia andenteritis. Postmortem examination of these animals revealed the presence of MPMV in thymus, lymph nodes,blood, brain, spleen, kidneys, and bone marrow based onelectron microscopy, induction of syncytia in indicator cellcultures, and immunofluorescence assays with MPMV anti-serum (39). Three characteristic hematological patternswere observed among the inoculated monkeys. One groupshowed no hematological aberrations, and less than one-third of the animals in this group survived. The remainderdied with symptoms indicative of runting or undue susceptibility to facultative pathogens. In these animals MPMVyielded symptoms indicative of a slow virus infection. Thesecond group exhibited basophilia with or without anemia,and none of these animals survived. The third group developed neutropenia and/or anemia and most survived (39). Inthese monkeys MPMV yielded symptoms indicative of anacute virus infection, eliciting hematological responsessuch as development of both MPMV-neutralizing and high-titered precipitating antibodies (31). The precipitating antibodies were directed against both envelope and internal(MPMV p27) antigens and persisted throughout the life ofthe animals suggesting that MPMV existed as a persistentinfection in these animals. Antibody-positive animals generally remained clinically asymptomatic throughout adultlife and showed no great variations in hematological patterns when compared with standards for hematologicaldata for control animals. Several MPMV-inoculated rhesusmonkeys of the third group succumbed several years afterinoculation to neurological syndromes characterized byloss of motor reflexes and limb paralysis (D. Fine and W.Loeb, unpublished data; A. Bogden, personal communication). Postmortem examination of one of these monkeysrevealed marked lymphadenopathy, and syncytia-inducingvirus was recovered from both lymph nodes and braintissues. Furthermore, a newborn rhesus monkey inoculatedwith a suspension of brain tissue from this animal developed a high-titered antibody to MPMV but showed nounusual clinical manifestations. Acute MPMV infections

may thus exhibit alternate clinical manifestations such asthose associated with other retroviruses. FeLV, for example, replicates in newborn cats and causes thymic atrophyin these animals (35). A runting disease characterized byweight loss and thymic atrophy has been reported in youngmice inoculated with Friend leukemia virus (98). Furthermore, inoculation of feral mice with a type C viruj indigenous to these mice induces a high frequency of either hindleg paralysis or lymphoma (43).

Although no oncogenic potential has been demonstratedfor purified MPMV, inoculation of MPMV-transformed RHFScells into newborn rhesus monkeys did induce palpablenodules of donor cell origin at the sites of inoculation.Microscopic examination of the nodules revealed a circumscribed area of proliferating undifferentiated or epithelialcells resembling those of the inocula. These cells weresurrounded by an infiltration of eosinophils, lymphocytes,and histiocytes, and there was fibroblastic activity aroundthe periphery. Pretreatment of inoculated monkeys withrhesus antilymphocyte serum enhanced both nodule sizeand duration of appearance (40). The nodules regressedwithin 2 to 3 weeks, after which the inoculated animalselicited high-titered MPMV-neutralizing and -precipitatingantibody (31). Expiant cultures derived from biopsied nodules released infectious MPMV and induced nodules whenreinoculated into other monkeys (40). Inoculation of MPMV-transformed RHFS cells into newborn rats and adultathymic nude mice (7, 39) also produced small nodules atthe site of inoculation, most of which regressed withinseveral weeks after inoculation. MPMV-transformed RHFScells resembled chemically transformed human embryo andforeskin cells in their ability to grow in soft agar and theirinability to produce malignant tumors derived from donorcells when inoculated into athymic nude mice (42). However, several athymic nude mice that had been inoculatedwith MPMV-transformed RHFS cells did develop invasivefibrosarcomas at the site of inoculation. Cytogenetic analysis of cell cultures derived from the respective fibrosarcomas showed the tumors to possess only mouse karyotype(39). Virus from these cell cultures did not induce tumors innude athymic mice, although the mice did elicit high-titeredprecipitating antibodies to both MMTV and MPMV. However, the cell cultures themselves were highly oncogenicand produced fibrosarcomas in nude athymic, C3H, andC3Hf mice within 2 weeks.

Viral Nucleic Acid Sequence Homologies

Two techniques of molecular hybridization have beenwidely utilized in examining the interviral relatedness of thetype D viruses. The first uses a radioactive DNA probe(cDNA) that is complementary to the viral RNA, and thesecond uses radioactive viral RNA as the probe. In the firsttechnique sequence homology between different viruses ismeasured by the degree of hybridization between the cDNAprobe and the RNA of other viruses. The second methodinvolves hybridization of radioactive viral 70S RNA to excessDNA from viral-infected cells that contain complementaryviral sequences. With this technique sequence homology ismeasured by the extent to which other viral RNA's can





compete for the ability of the radioactive RNA probe tohybridize to the cellular DNA.

According to the competitive hybridization techniquewith radioactive MPMV 70S RNA (25), MPMV was indistinguishable from X-381, AO, and the HeLa isolate of Bauer

(10) and only partially related (approximately 30%) to theendogenous langur virus, PO-1-Lu (12). No sequence ho-mology was observed between MPMV and the RNA's of

SSV, MMTV, murine leukemia virus, B-GPV, AMV (25), and

SMRV(26,41,91).With the use of virtually complete [3H]cDNA transcripts of

PO-1-Lu, approximately 22% nucleic acid sequence homol-

ogy was detected with MPMV 70S RNA (12). In reciprocalexperiments, MPMV [3H]DNA transcripts hybridized approximately 30% to the PO-1-Lu genome (12). In the samestudies the PO-1-Lu genome was reported to be distinct

from other retroviruses, including GALV, SSV, and theendogenous viruses of Mus cervicolor (M432, CERV CI, andCERV Cil), Mus musculus (BALB/c xenotropic), and pig(CCL33) origin. A low degree of hybridization (4 to 5%) wasseen with the RNA of an endogenous baboon virus (M28),RD114, and the squirrel monkey virus isolate, M534 (12).The significance of this low level of hybridization is unclearbecause it is not known whether it represents specificsequence homology or higher background hybridizationvalues. No sequence homology has been observed betweenSMRV, MPMV, and AMV. These results support the conclusion that PO-1-Lu is a new viral isolate of Old World

monkeys that shares significant sequence homology withMPMV.

On the basis of direct hybridization of [3H]SMRV 70S RNAto the DNA of MPMV-producing cells and competitionhybridization with [3H]SMRV 70S RNA, no sequence homol

ogy was observed between SMRV, MPMV, and AMV (26, 41,91). Direct hybridizations with the use of cDNA of SMRV toRNA's of MPMV, baboon leukemia virus, SSV, and GALV

also revealed no sequence homology with SMRV (54).Natural Distribution of Viral Nucleic Acid Sequences. By

definition endogenous virogenes are those sets of genesequences that form an integral part of the chromosomalDNA and that can, under certain conditions, code for theproduction of a retrovirus. A species of origin has beenreported for essentially all type B and type C endogenousviral gene sequences in 1 or more animal hosts. MPMV,although originally isolated from a rhesus monkey (M.mulatta), until recently remained a virus in search of aspecies containing representative endogenous viral genesequences. With direct hybridization of radioactive MPMV70S RNA to DNA of various animal species, it has beenshown that a portion (approximately 20%) of the MPMVgenome was present in the cellular DNA of several OldWorld monkeys of the subfamily Cercopithecinae (34). Nosequence homology was observed between MPMV and thecellular DNA's of New World monkeys, apes (including

man), and several nonprimates (Ref. 34; Table 4). Thisobservation has been confirmed and extended with the useof essentially complete cDNA transcripts of the MPMVgenome (12). The data obtained with the use of cDNAprobes show that MPMV exhibits considerably more nucleicacid sequence homology to the cellular DNA of members ofthe Colobinae subfamily than to members of the Cercopi-

Table 4Natural distribution of type D retrovirus proviral sequences in the

DNA's of various animal species"

SpeciesMPMVÂ»PrimatesOld

WorldmonkeysColobinaeLangur

+ +cColobus

++CercopithecinaeBaboon

+Mangabey+Macaques+Celebus

apes+Quenon+Patas

+ApesGibbonChimpanzeeManNew

WorldmonkeysSquirrelmonkeyWoollymonkeyCapuchinNTMarmosetHowlerCebusSaki

NTSpidermonkeyNTOwl

monkeyPo-1-Lu+

+ +++++++++++â€”-â€”--NTNTNTNTNTNTNTSMRVNTNTNTNTâ€”NTNT-â€”-â€”+

+ ++-â€”â€”NTNT--â€”

NonprimatesCowRabbitHippopotamusSheepPigDogCatMouseRatChickenHamsterGuinea pig

NTNTNTNTNTNTNTNTNT

NTNTNTNT

NTNTNTNT

" Data tabulated from Refs. 12, 25, 26, 34, 41, and 91.* Includes X381, FTP-1, and HeLa isolates (indistinguishable

from MPMV based on hybridization and immunoassay criteria).c + + + +, complete homology, + + +, high degree of homology;

+ + , partial homology; +, low homology; -, no homology; NT, nottested.

thecinae (32 to 45% versus 13 to 16%, respectively) (12).The C0f,/2's (20 to 50 mol x sec/liter) for both subfamilies

indicate that these sequences were present in multiplecopies (20 to 50) as compared to a C,,f,/2 of 900 mol x sec/liter in MPMV-infected A204 cells (12). The presence of

multiple copies has been considered a good criterion fordemonstrating endogenous sequences in host cell DNA, inthis case endogenous sequences in Old World monkeys.Because the highest final extent of homology (45%) wasobtained with the spectacled langur (P. obscuris) DNA ascompared with colobus (Colobus guereza) DNA (32%), ithas been suggested that MPMV was derived from an endogenous virus of one of the Asian rather than African generaand, perhaps, from the spectacled langur specifically (12).

Essentially, full-length cDNA probes of PO-1-Lu hybrid

ized completely to DNA of a variety of spectacled langurmonkey tissues with a C0f,/2 of approximately 50, consistent

OCTOBER 1978 3131




with the presence of multiple copies (20 to 40 per haploidgenome) of the PO-1-Lu genome (12). The presence ofmultiple copies of viral sequences in langur cellular DMAindicates that PO-1-Lu is an endogenous virus of spectacledlangur monkeys. The same probe hybridizes 75% to Pres-bytis senex (purple-faced langur) and 59% to C. guereza, amember of the Colobinae subfamily, and from 12 to 15% tothe cellular DNA's of 10 different species belonging to 6genera of the Cercopithecinae subfamily (12). The C0f1/2's

for these sequences also indicate their presence in multiplecopies. No sequence homology was observed between thePO-1-Lu cDNA probe and the cellular DNA's of New World

monkeys, apes, including man, and several nonprimates(12).

Using direct hybridization radioactive SMRV 70S RNAhybridized significantly (52 to 77%) to the DNA's of a variety

of squirrel monkey tissues (26, 41, 91). The kinetics ofhybridization yielded C0f,/2'sof 120 to 550 (26, 91), consist

ent with the presence of the SMRV genome in multiplecopies. The presence of multiple copies of viral sequencesin squirrel monkey cellular DNA indicates that this virus isendogenous to S. sciureus. No sequence homology wasobserved between the SMRV 70S RNA probes and thecellular DNA's of other New World monkeys, Old World

monkeys, apes (including man), and several nonprimates(91). Similar results were reported with cDNA probes ofSMRV (13, 54).' In contrast to the wide distribution of

proviral sequences of BaEV, PO-1-Lu, and MPMV in OldWorld monkeys, SMRV proviral sequences, even under low-stringency hybridization conditions (13), have been identified only in squirrel monkeys and not in other New Worldmonkeys that share habitats. The absence of SMRV proviralsequences in other New World monkeys may indicate ahigh degree of divergence among these animals makingdetection of these sequences difficult. Alternatively, SMRVproviral sequences may have been acquired by squirrelmonkeys after their divergence from other New Worldmonkeys. An interesting observation has been reportedrecently (13) demonstrating the presence of gene sequences partially homologous to SMRV in the cellular DNAof normal tissues of the New World carnivore, the skunk(Mephitis mephitis and Spi/ogale putorius). The final extentof hybridization of SMRV cDNA to skunk DNA was 8 to 10%under relatively stringent hybridization conditions and 15 to17% under lower stringency conditions, whereas 100%hybridization was achieved to squirrel monkey DNA undereither conditions. No sequence homology was observedwith other members of the family Mustelidae (mink andweasel), as well as other New World carnivores, includingracoons and various cats (13).

Immunology

Evidence for the uniqueness of MPMV, in comparison toother members of the retrovirus genera, was originallyderived from immunological studies of the virus in whichimmunodiffusion was used (5, 77). The development ofsensitive RIA's for the virion structural proteins p27, p15,

and gp70 has greatly expanded our understanding of therelatedness of MPMV to other retroviruses. Table 5 presentsa summary of results for the homologous assays for these 3

proteins. On the basis of these assays, MPMV exhibited nocross-reactivity with type B and type C retroviruses ofprimate (SSV, GALV, BaEV), feline (RD114, FeLV), rodent(murine leukemia virus, MMTV, B-GPV, pig virus, CCL38),and avian (AMV, RAV-2) origins. In addition, no cross-reactivity was observed between MPMV and members ofthe genera Lentavirus (including Visna and maedi virus)and Spumavirus (including the simian syncytial or so-calledfoamy virus) (see Ref. 30 for retrovirus classification). Nordoes MPMV cross-react with BLV and EIAV, 2 as yetunclassified retroviruses. In contrast, the X-381, FTP-1, andAO isolates are immunologically indistinguishable fromMPMV based on p27 RIA's (Table 5). X-381 and FTP-1 are

also indistinguishable from MPMV in a homologous RIA forMPMV gp70 (Table 5). With the less sensitive technique ofimmunodiffusion, the HeLa cell isolate (10) was shown tohave cross-reactivity with MPMV but not with F-MuLV,FeLV, and MMTV. The cross-reactivity between MPMV andHeLa virus was supported by use of indirect immunoferritinlabeling in which antisera raised against the respectiveviruses revealed cross-reacting antigenic specificities onthe surfaces of both HeLa virus and MPMV. Cross-absorption and end-point dilution techniques also indicated thepresence of similar if not identical envelope antigens (45).Similarly, with the use of RIA's for MPMV p27, the isolate

from J-96 cells has been reported by 2 laboratories topossess an antigen identical with MPMV p27 (18, 81),whereas a third laboratory reported the J-96 isolate topossess an antigen similar to but not identical with theMPMV p27 (106). Because the J-96 cells possess HeLa cellmarkers (75) and because all viral isolates from HeLa orsuspected HeLa cells have thus far been shown to beidentical with MPMV, further studies on the J-96 isolate mayshow this virus to be identical with MPMV.

Immunological analysis of viral RT also provides amethod for classifying retroviruses into distinct groups.Antiserum prepared against partially purified RT of MPMVwas found to neutralize the endogenous polymerase activityof MPMV (105), X-381 (105), and AO (81) viruses. Theantiserum did not inhibit the RT of AMV, FeLV, R-MuLV, F-MuLV, SSV, and MMTV (81, 105).

The antigenic properties of the langur monkey isolate,PO-1-Lu, have been compared with those of MPMV afterhomologous RIA's with the use of purified MPMV proteinsp27 and gp70 and antiserum to MPMV (99). PO-1-Lu contained an antigen that was antigenically indistinguishablefrom MPMV p27. In contrast PO-1-Lu failed to compete inthe assay for the MPMV envelope protein gp70. PO-1-Lualso has been reported not to exhibit cross-reactivity inRIA's for the major structural proteins of any of the known

type B and type C retroviruses (99).In contrast to PO-1-Lu, the squirrel monkey isolates

SMRV and M534 exhibited no antigenic cross-reactivity inhomologous RIA's for both MPMV p27 and gp70 structuralproteins (41, 92, 99). Initial studies with both species- andinterspecies-specific immunodiffusion assays revealed noantigenic relatedness between SMRV and MPMV, MMTV,BaEV, SSV, R-MuLV, RD114, BLV, EIAV, FeLV, GALV (92),and B-GPV.4 More sensitive interspecies RIA's have been

reported that do detect cross-reactivities between MPMV,BaEV, RD114, SMRV, and PO-1-Lu. With the use of a broad





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

Homologous RIA for MPMV structural proteins

Interspecies RIA

VirusType

DMPMVX-381FTP-1AOJ96PO-1-LuSMRVM534Type

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

BMMTVB-GPVType

EVisna'"l-labeled

'"l-labeled

p27p15+

6++

ND+rND+

ND+ND+NDNDNDND_

_â€”_-_-_-_â€”_-

_NDNDNDNDNDNDND

NDNDNDâ€”

â€”¿�ND-e

ND'Â«l-labeled

gp70++

c+rNDND-_-ND_ND____NDNDND_NDNDNDNDNDNDNDAnti-MPMV

and '"l-la-

beled BaEV9P70++

c+rNDND++

f+ND__ND_+__NDNDND+NDNDNDNDNDNDNDAnti-MPMV

p27 and 125I-

labeledSMRVp35++

c+rNDND++NDND_NDND____NDâ€”ND_NDâ€”_-â€”NDNDAnti-MPMV

and I25l-la-

beledSMRVgp70+NDNDNDNDND+NDND_NDND_+__NDNDND+ND_NDND_NDND

â€¢¿�Compiled from 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 MPMVand 125l-labeled BaEV gp70, a cross-reactivity was reported

between MPMV and the 2 type C retroviruses, BaEV andRD114 (96). This reactivity did not extend to other type Cviruses, GaLV, SSV, FeLV, R-MuLV, and AKR murine leukemia virus (96). Subsequent studies with this broad interspecies assay have demonstrated that PO-1-Lu and SMRValso share cross-reactivity with the gp70's of MPMV, BaEV,and RD114 (99). However, interspecies RIA's for SMRV p35

and MPMV p27, with both goat and rabbit antisera to SMRVand MPMV, demonstrated cross-reactivity only betweenMPMV, SMRV, and PO-1-Lu (28, 31, 54). No cross-reactivitywas observed in these assays for the pSO's of mammalian

type C (SSV, BaEV, RD114) or type B (MMTV, BLV) retroviruses (28, 31, 54). Conversely, in interspecies assays forpSO's of mammalian type C viruses, MPMV shares no

detectable cross-reactivity (82). These findings are significant, especially in view of the recent demonstration (31, 36)

that natural sera from both Old and New World monkeyscontain antibodies that recognize the retroviruses derivedfrom that species (e.g., SMRV and MPMV) and also exhibitinterspecies cross-reactivities with MPMV, BaEV, SMRV,and RD114. Table 6 summarizes the species and interspecies reactivities determined with natural sera from prosimi-ans, Old World monkeys, New World monkeys, and apes. Itis evident that in some but not all of the animals tested thusfar, naturally occurring antibodies recognize specific determinants of the major nonglycoproteins shared by the typeD retroviruses of both Old World (MPMV) and New World(SMRV) monkeys, but not with the p30 of BaEV. Initialindications that similar immunological reactivities also resided with the envelope proteins of these viruses, in addition to BaEV, were suggested by the demonstration thatnatural sera cross-reacted with MPMV, SMRV, and BaEVwith the use of intact virus competition RIA's (41). A subse

quent study (32) in which purified envelope glycoproteins

OCTOBER 1978 3133




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

MPMVSMRVProsimians

Bush babyOrigin

ofsera(Ga/ago

crassi-caudatus)Whole

virus0/13p27 Whole virusp350/13BAEVWhole virus p30

Old World monkeysMacaque

Grivets

Baboon

Mangabey

New World monkeys

(M. mulatta)

(Macaca fascicu-laris)

(Macaca speciosa)

(Cercopithecusaethiops)

(Pap/o cynoceph-alus)

(Pap/o anubis)(Pap/o hamad-

ryas)(Cercocebus atys)

20/121" (40-20.250)''

6/21 (400-51,200)

0/10

3/19

7/15(40-20.250)

1/110/2

25/121 (20-10,240)

1/2(2,560) 3/19

1/10(400)

0/10

3/19

7/15(40-20.250)

1/111/2

0/2

5/121 (20-80)

0/2

0/10

0/75

0/2

7/10(200-1,600) 0/10

SquirrelCapuchinOwlMarmosetSpiderHowlerApesChimpanzeeGorillaOrangutan(S.sciureus)(Cebus

capuci-nus)(A.

trivirgatus)(Saguinusoedi-pus)(Saguinus

fusicol-lis)(Saguinus

nigri-collis)(Callithrix

jac-chus)(Ã•teles

sp.)(A.belzebÃ¹!)(P.

troglodytes)(Gorillagorilla)(Pongo

pygma-cus)14/122

(>40)0/140/60/20/10

0/53/12

(100-200)1/3(100)0/60/60/719/122(250-

2/14(40-80)2,050)2/6

(250-750)0/23/9

0/45/12

(100-200)1/31/60/60/70/30/70/40/120/5

" 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 hyperimmunesera were used demonstrated that the interspecies reactivityobserved with intact virus resided at least in part with thisantigen. The findings with the natural sera are in agreementwith those studies in which hyperimmune sera directedagainst their respective viruses were used. The prevalenceof antibodies cross-reactive with type D retroviruses isevident from the fact that they have been found in newlyimported rhesus monkeys as well as in rhesus monkeysbred in captivity in geographically separated primate centers throughout the United States.

Evolutionary Prospectus for Type D Viruses in Primates

Based on the cross-reactivity observed between the majornonglycoproteins of the distantly related primate endogenous retroviruses of langur and squirrel monkeys (28), it is

possible that these determinants represent highly conserved amino acid sequences that were present in a progenitor virus common to all primate type D retroviruses eventhough they share no detectable nucleic acid sequencehomology. This progenitor type D virus would have to havebeen integrated within the genome of an ancestor commonto both Old and New World monkeys prior to their divergence approximately 50 million years ago (Chart 1). Furthermore, this progenitor virus would have been distinctfrom the progenitor virus of the endogenous type C primateretroviruses because of the lack of any cross-reactivity ofthe BaEV p30 with the analogous proteins of PO-1-Lu,SMRV, or MPMV. Evidence for an earlier common progenitor virus for both type C and type D retroviruses could beargued from the observations of interspecies reactivities ofthe envelope glycoproteins of PO-1-Lu, SMRV, and nowBaEV. Conservation of the 3-dimensional structures under-





Primate Group SubfamilyEndogenous Exogenous

Genus Virus Virus Immunological AssayGroup Interspecies Interspecies

p2T-p55 gpTÃŒT

New World

Type D VirusIntegration

I Alouatta

ICebus

I Aolus

l Lagothrix

Â»SaimirÃ - SMRV

Gorilla

Pan

Homo

Pongo

Hylobates

Presbytls - Po-1-Lu

Colobus

Erylhrocebus

Cercopithecus

Cercocebus

Macaca

MandrillusPapio BaEV

50 40 10 030 20Millions of Years

Chart 1. Evolution of type D retroviruses of Old and New World monkeys based on immunological cross-reactivities (see "Immunology"),

reactivities between endogenous viruses; . additional cross-reactivities between the endogenous viruses and the exogenous virus, MPMV.-, cross-

lying these reactions may have enabled the viruses to gaina selective advantage for survival either by protection fromimmunological surveillance by a host carrying an endogenous virus with similar sequences or by the ability of thevirus to infect evolutionary descendents derived from thehost carrying the earliest common progenitor virus. Evidence supporting the latter hypothesis may derive from thereported ability of RD114 (which was derived from BaEV)gp70 to block MPMV infectivity in KC cells, suggesting thatthese viruses share common receptor sites (see "Infectivity"). This observation is important in light of the cross-reactivity between gp70's of RD114 and BaEV (96). Therefore, one might also predict that MPMV, PO-1-Lu, SMRV,BaEV, and RD114 all share common determinants that arepossibly involved in adsorption to specific target cells andthat are also the same determinants involved in the inter-species cross-reactivity between their viral glycoproteins.However, we cannot rule out the possibility that the cross-reactivities of the envelope proteins were derived from arecombination of sequences coding for the gp70's of a type

D (e.g., PO-1-Lu) and a type C (e.g., BaEV) retrovirus. Thisevent would confer the same selective advantages as thosethat might have arisen by the evolutionary conservationprocess discussed above. The demonstration of these inter-species cross-reactivities that are shared by distantly related primate retroviruses, particularly those associatedwith major envelope proteins, provides a means for detecting determinants that are representative of all primateretroviruses presently known and yet to be isolated. Thus,these assays may provide useful tools for the detection ifnot the isolation of a human retrovirus.

Type D Viruses in Humans

The majority of the studies published on the presence oftype D viruses in humans have been concerned primarilywith MPMV and its role in human neoplasia, particularlymammary neoplasia. The numerous attempts to implicateMPMV in the etiology of mammary neoplasia are obviously

derived from the fact that MPMV was initially isolated froma spontaneous mammary tumor of a nonhuman primate.These studies have attempted to provide evidence for thepresence of whole virus or viral components (proteins and/or nucleic acids) in human breast tumors. Although manyof the studies have indicated the presence of viral components, none of the individual positive findings have beenreproduced in other laboratories. The various reports ofpositive findings of MPMV in fresh human material (ascompared to continuous cultured cell lines) can be summarized as follows.

1. Sequence homology was reported between MPMV[3H]cDNA and RNA from human malignant breast tu

mors (27). In the same studies no sequence homologywas observed with the same probe and RNA of benignbreast tumors or normal tissues. However, sequencehomology was assayed by hydroxylapatite chromatog-raphy and cesium sulfate centrifugation. These assaytechniques could artificially yield high degrees ofhybridization as compared to analysis by S, nucleasedigestion. In fact, with the S, technique, no sequencehomology was observed between MPMV [3H]cDNAand RNA of human malignant breast tumors (102).Further studies with MPMV cDNA probes representingthe entire viral genome and in which alternate approaches are used to detect hybrid formation will berequired to resolve these discrepancies.

2. With the use of a sensitive RIA for MPMV p27, anti-genie reactivity related to this protein was reported in8 of 18 partially purified preparations derived fromhuman breast tumors (107). In the same study noreactivity was detected in normal placental tissues orin tumors that were not of breast origin. In contrast asimilar study (18) with analogous procedures reportedno MPMV p27 reactivity in 15 breast carcinomas examined. The second study used individual columnsfor the purification of cross-reactive antigens fromeach tumor to eliminate any residual antigen carryoverfrom virus purification. A protease inhibitor was also

OCTOBER 1978 3135




included in their RIA buffers to eliminate false-positivereactions due to proteolytic activity. Negative findingswere also reported with the less sensitive technique ofimmunodiffusion (77).

3. Antibodies reactive with 125l-labeled MPMV p27 were

detected in plasma from patients with various cancers,including acute myelogenous leukemia and breastcancer, as well as from individuals without evidenceof cancer (63). These results were not substantiated in2 independent studies (18, 31). In the latter 2 studies,assays were performed on a combined total of 240sera collected from individuals with various cancers(including 54 with breast cancer), with other disorders,and with no diagnosed disease.

4. Ohno ef al. (79) reported the immunological cross-reactivity of RT from MPMV with a Mg2'-preferring

DMA polymerase previously purified from humanbreast cancer particles (80). Cross-reactivity was observed by both inhibition of enzyme activity and complex formation between the purified human particleenzyme and anti-MPMV RT IgG. No such interactionswere observed with RT's of AMV, R-MuLV, F-MuLV,

FeLV, SSV, and MMTV (79). In the same studies, DNApolymerase-containing particles isolated from spleensof chronic myelogenous leukemia, chronic lympho-cytic leukemia, and Hodgkin's disease showed no

cross-reactivity with anti-MPMV RT IgG. Although theassociation of this antigenic reactivity with breasttumors has potential applications as a diagnostic tool,currently the assay is cumbersome and lacks sensitivity and reproducibility from specimen to specimen.

5. Particles resembling MPMV have been observed byelectron microscopy in negatively stained preparations of human milk concentrates (20, 88). In thesecombined studies 841 milk samples collected from 723women with and without familial history of breast orother types of cancer were examined for the presenceof particles morphologically resembling MPMV. Although MPMV-like particles were observed in 184 ofthe samples, no differences were observed betweenthe incidence of occurrence of these particles in milksfrom women with familial history of breast or othertypes of cancer and those from women without such ahistory. At present the actual identity of such milk-associated MPMV-like particles is unclear since theyhave no reported biological activity (e.g., infectivityfor cell cultures) and thus may represent cell fragments.

These conflicting studies on the association of MPMVwith human neoplasia indicate the need for further studieswith new and more sensitive techniques. The broad inter-species assays (28, 32, 36, 41, 54, 96, 99) initially developedto identify immunological cross-reactivities between themajor glycoproteins of type D viruses and other distantlyrelated primate retroviruses such as type C viruses mayprovide the most sensitive test systems for detection ofhuman retroviruses.

Addendum

Subsequent to submission of this review, two studies

presented data extending our knowledge of MPMV. Thefirst study (W. Drohan, D. Colcher, and J. Schlom. The Useof Standard and Relaxed Hybridization Conditions to DetectTwo Classes of Sequences Related to Type D Retrovirusesin the DNA's of Primates. Biochim. Biophys. Acta, in press)

demonstrated the existence of two classes of MPMV-relatedsequences in the DNA's of various Old World monkeys. One

class consists of the previously reported (34) sequenceshighly related to about 20% of the MPMV genome. Thesequences in the second class are related to an additional20 to 40% of the MPMV genome and are detected onlyunder low stringency conditions of nucleic acid hybridization. These latter sequences represent highly divergedMPMV sequences as evidenced by the low thermal stabilityof the hybrid duplexes and provide evidence that numeroustype D viruses exist in the primate population. The secondstudy (D. Colcher, Y. A. Teramoto, and J. Schlom. Immunological and Structural Relationships between Langur Virusand Other Primate Type D Retroviruses. Virology, in press)demonstrates that although MPMV and PO-1-Lu cannot bedistinguished by homologous RIA's for their p27 structural

protein, they can be readily distinguished in a RIA for theirp10-p12 proteins. This provides an additional type-specificassay to distinguish between the two viruses.

Acknowledgments

We wish to thank our colleagues. Dr. R. Gilden, Dr. H. Rabin, Dr. L.Arthur, and Dr. R. Massey, for critical evaluation and suggestions and M. A.Gonda and M. Gregg for preparation of electron micrographs. We aregrateful to M. Loose for considerable assistance in the technical preparationand editing of the manuscript. We also wish to acknowledge P. McLane, L.Conners, and J. Clarke for help in typing the manuscript.

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Fig. 1. Comparative morphology of type B, C, and Dretroviruses. a, cluster of Â¡ntracytoplasmic type A particles (representing immature precursor of budding typeB virus) with doughnut-shaped nucleoid and electron-lucent center; Ã¹, immature type B virus with doughnut-shaped nucleoid and electron-lucent center buddingfrom cytoplasmic membrane; c, mature, extracellulartype B virus with eccentric electron-dense nucleoid andcharacteristic surface spikes; d, lack of morphologicallyrecognizable Â¡ntracytoplasmic form for type C virus, e,immature type C virus with crescent-shaped nucleoidbudding from cytoplasmic membrane; f, mature, extracellular type C virus with centrally located condensednucleoid; g, intracytoplasmic type A particle with electron-dense, doughnut-shaped nucleoid and projectionson the outer perimeter of the nucleoid (representingimmature intracytoplasmic precursor form of type Dvirus); h, immature type D virus with doughnut-shapednucleoid with electron-lucent center budding from cytoplasmic membrane; I, mature extracellular type D viruswith condensed bar-shaped nucleoid. All micrographsare approximately x 87,000. Thin sections were double-stained with uranyl acetate and lead citrate. Type Bviruses were MMTV from C3H/Cgrl mammary adenocar-cinoma cell cultures. Type C viruses were Theilen strainof FeLV cultivated in FL74 feline lymphocytes. Type Dviruses were MPMV grown in A204 cultures.

\9-

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OCTOBER 1978 3139



1978;38:3123-3139. Cancer Res Donald Fine and Gerald Schochetman Type D Primate Retroviruses: A Review

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