Interspecies antigenic determinants of the reverse transcriptases and p30 proteins of mammalian type C viruses.

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JOURNALOFVIROLOGY,June1975,p.1440-1448

Vol. 15,No. 6 Printed inU.S.A.

Interspecies Antigenic Determinants

of the

Reverse

Transcriptases and p30 Proteins

of Mammalian

Type

C

Viruses

CHARLES J. SHERR,* LOUIS A. FEDELE, RAOUL E. BENVENISTE, AND GEORGE J. TODARO

Viral Leukemia andLymphoma Branch, National Cancer Institute,Bethesda, Maryland20014

Received forpublication 7 February 1975

The major internal structural proteins (p30) of type C viruses isolated from

several mammalian species were studied by radioimmunoprecipitation and

competitive radioimmunoassays. Three antigenically distinguishable sets of interspecies determinants could be demonstrated by both methods. One setof

determinants sharedbyviruses of rodentorigin (mouseandrat) canbedetected

readily in feline leukemia viruses butnot in othertype C viral groups. The p30 proteins ol murine viruses also contain a second discrete set of antigenic

determinants related to those in infectious primate viruses and endogenous

porcine viruses, but not detected in the feline leukemia virus group. The p30 proteinsofendogenousviruses of baboons and domesticcatsshareyetathirdset

ofcross-reactive determinantsnotdetected in type C viruses isolated from other species of animals. Enzyme inhibition studies performed with antisera raised

toward thereverse transcriptases of thesesame groups oftypeC viruses showed

the same patterns of immunological cross-reactions as observed with p30

pro-teins. The antigenic cross-reactions between the homologous proteins oftype C

virus isolated from genetically distant animals mayreflect transmission oftype

Cvirusesacrossspecies barriers.

Sensitive

immunological

assaysfor the struc-tural proteins of mammalian type C viruses have becomeincreasinglyimportant in

classify-ing new viral isolates and relating them to

known groups of viruses. Agroup-specific (gs-1) antigenicdeterminantshared

by

murine typeC viruses was first described

by Gregoriades

and

Old (17) and has been

assigned

to a protein of

approximately

30,000 molecular

weight (p30),

themajor internalproteinofthe virion(32, 42). Subsequent studies showed that the p30 pro-teins of murine type C viruses also share an

interspecies antigenic determinant (gs-3) with

other rodent type C viruses (rat and hamster)

and with the feline leukemia viruses (11, 13, 33,

35, 43). With the characterization of type C

virusesisolatedfromother mammalian species, it hasbecome apparent that thep30proteinsof certain groups ofisolates show only weak gs-3 reactivity, whereas other classesofinterspecies antigenic determinants can be demonstrated (14, 14a, 21, 31, 35, 36, 44, 48). The immuno-logical cross-reactions observed between dif-ferent groups of viral p30 proteins do not ap-pear to be fortuitous, since similar cross-reac-tions are observed with other viral components including reverse transcriptase (1, 26, 37) and theenvelopeglycoproteingp7O(51, 52).

Serological and amino acid sequencing

stud-ies of homologous proteins from different

spe-cies have demonstrated a correlation between

the extent of antigenic cross-reaction and the

degree of sequence homology. Such approaches canbe used to estimate theextentof

evolution-ary divergence between species and provide dataingeneral agreement with taxonomic

clas-sifications based on anatomical considerations

and the fossil record (8, 15, 19, 20, 23, 28, 411. Similar experiments have indicated that the immunological cross-reactions detected

be-tween homologous proteins of different groups

of type C viruses are due to similarities in the amino acid sequences ofthese proteins as well (12, 34). Thus, the antigenic relatedness of the

structural proteins of different type C viruses

might reflecttheir ancestral origins and evolu-tionary history in different species of animals (12, 47).

The present report describes studies of in-terspecies antigenic determinants cn the p30 proteins and reverse transcriptases of several groups ofmammalian type C viruses. The data show that at least three antigenically distin-guishable classes of interspecies determinants can be detected among the groups of viruses studied. We propose that homologies observed between type C viruses isolated from genetically

distant vertebrate species reflect trans-species infections of either somatic cells, germ cells, or

both, which have occurred in the past (7).

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MATERIALS AND METHODS

Viruses and cell lines. The mammalian type C

virusesused inthese studies are listed in Table 1 and

weregrown as described previously (49). Viruses were

concentrated from culture supernatants by

continu-ous-flowcentrifugation and were banded on sucrose at

adensity of 1.15 to 1.17g/cm3.

Purification of viral proteins. The p30 proteins of the simian sarcoma-associated virus (SSAV), the gibbon ape leukemia virus (GALV), the endogenous baboon virus M7, the endogenous domestic cat virus RD-114, the endogenous mouse virus S2CL3, feline leukemia virus (FeLV, Rickard strain), the murine leukemia virus (MuLV, Rauscher strain) were purified

by gel filtration and isoelectric focusing (49). The

p30protein of the endogenous porcine PK(15) virus

waspurifiedby column chromatographic methods as

described by Strand and August (51). All proteins had approximate molecular weights of 30,000 as

de-termined by electrophoresis in 7 and 13%

poly-acrylamide gels containing sodium dodecyl sulfate, and all were greater than 95% homogeneous as judged by multiple electrophoretic criteria (49). Viral reverse

transcriptases were partially purified as described

previously (40, 46).

Antisera. Antiseratop30proteinsand viral

polym-erases wereraised in New Zealand white rabbits (37,

46, 49). Animals received primary immunizations in

complete Freund adjuvant divided between the four footpads and two intradermal booster injections in

incomplete adjuvant. Intramuscular immunizations

were continued at 3-week intervals, and testbleeds

were obtained from an ear artery 7 days after each

booster injection.

Groups of two to three animals were immunized

with the same antigen. By the schedule described

above,maximumantibody titers top30proteins were

routinely obtained after four to five immunizations. Sera obtained at this time were titered by

immuno-precipitation withhomologous andheterologous

radio-labeledp30 proteins. No significantdifferenceswere

observed in the relativetiters of cross-reacting

anti-bodiesinindividual sera from eachgroup of animals.

Antisera to certain viral polymerases (RD-114, MuLV, FeLV) were raised in single rabbits, whereas

sera directed toward other viral enzymes [SSAV,

GALV, M7, PK(15)] were raised in groups of two to

three animals. In cases where comparisons were

possible, sera from different animals showed the same

patterns of cross-reactivity when used to inhibit

enzymes from heterologous viruses.

TABLE 1. Mammalian type C viruses studied

Isolatedfrom: Virus Characterization

MuLV(Rauscher strain) (38)

S2CL3 (55)

R4/B(4) S16CL10(I) (4)

AT-124(56)

RT21C(53)

CCL 38(25)

V-NRK (25)

FeLV(Rickardstrain)(39)

FeLV-FSV(Gardner-Arnstein

strain)(10)

RD-114(30)

CCC (9,27)

PK(15) (57)

MPKa

M7(3)

M28(59)

SSAV(54)

GALV(24)

NB-tropic leukemogenic virus propagated in BALB/c

JLSV9 cells

N-tropic virus spontaneously released from BALB/c

S2CL3cell line

B-tropic virus releasedfromBALB/cR4/Bcell line

Xenotropic virus induced from BALB/c cell line and

propagatedinrabbitSIRC cells

Xenotropic virus recovered from human RD cells

pas-sagedinimmunosuppressedNIHSwissmice

Endogenous rat virus released from cultured rat thymus

cells

Rat virus released from cultured Walker rat carcinoma

cells

Endogenous rat virus spontaneously released from rat

NRKcells

Feline leukemia viruspropagatedin F422catcells

Feline leukemia/sarcoma virus propagated in FCf2Th

caninethymuscells

Endogenouscatviruspropagatedinhuman RD cells Endogenous cat virus spontaneously released by feline

CCC cells

Endogenous porcine virus spontaneously released

by

pigkidneycells

Endogenous porcine virus spontaneously released from

minipigkidney cells

Endogenous baboon virus isolated from baboon placenta

andpropagatedinrhesusmonkeycells

Endogenous baboon virus isolated from cultured baboon

cells and growninhumanA204cells

Simian sarcoma-associated virus isolated from a

woolly

monkey fibrosarcoma andgrowninhumanA204cells

Leukemia virus isolated from a gibbon lymphosarcoma

and growninhumanNC37 cells

aM.M.Lieber,C. J.Sherr,R. E.Benveniste,andG.J.Todaro, Virology,inpress.

Mouse

Rat

Domesticcat

Pig

Primates

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SHERR ET AL.

Radioiodination. Purified p30 proteins (10 gg)

were radiolabeled with 125I by the chloramine T

method to approximately equivalent specific activi-ties (5

mCi/mg)

(16).Labeledtest antigenswere stored in 0.05 M phosphate buffer (pH 7.5) containing 1%

bovine serum albumin and were used within 8 days

afterlabeling. More than90% ofeach labeledantigen

could be specifically precipitated bythe homologous

antiserum.

Radioimmunoassays. Antisera titrations and

com-petitive radioimmunoassays were performed by a

"double-antibody" method (35, 46). Competition

as-says were initiated with 50% of the iodinated test antigen bound, anddetergent-disrupted viruses were used ascompetingantigens.Immunecomplexeswere precipitated with a titered excess of anti-rabbit 7S

globulin raised in sheep (Pocono Rabbit Farm and

Laboratories, Canadensis, Pa.). Supernatants were

counted by liquid scintillation to a +3% error, and

counting efficiency was monitored by external

stan-dardization.Thedataareplottedaspercent

displace-ment of the test antigen from immune complexes

(ordinate) versus micrograms of competing protein

(abscissa). Pointsonthe competitioncurvesrepresent

the average ofthree to six determinations for each

competingantigen (standarddeviation < 8%). Inthe

assays shown, no significant antigenic differences

wereobserved between the p30proteins ofthesame

typeCviruspropagatedincellsfromdifferentspecies

or between different isolates from the same type C

viral group(cf.Table 3).

Polymerase inhibition studies. All studies were

performed with immunoglobulin G (IgG) purified

from sera by salt fractionation and chromatography

onDEAE-cellulose.Reversetranscriptaseassayswere

performed,asdescribedpreviously (37), witha

polyri-boadenylate templateandanoligodeoxythymidylate

primer. Reactions were initiated with a mixture of

template, primer, and substrate.Detergent-disrupted

viruses(0.01 ml)wereusedas a sourceofenzyme,and

IgG was added prior to the addition ofenzyme. At

least 70,000 counts of [3H]thymidine

5'-monophos-phate per min wereincorporatedinto

acid-precipita-ble polydeoxythymidylate product after 60 min at

37C in theabsence ofimmune

IgG.

Incorporation

of

[3H]thymidine 5'-triphosphate waslinear for 60 min

intheabsenceofspecific antibodies. Thequantitiesof

immune IgG required for 30% inhibition ofenzyme

activitywerecalculatedfrominhibitioncurves

devel-oped with serial dilutions of antibody. The actual

amounts ofIgGrequired for these levelsofinhibition

in homologous antibody-antigen systems are

pre-sented inthe legend to Table 4. The data havebeen

normalizedby assigninga value of 1.0 for the homolo-gous system.

Proteindetermination.The concentrations of pu-rified rabbit IgG were determined from absorbance

measurements at 280 nm (E"'-m = 14). All other

proteins were quantitated bythe method ofLowry et

al. (29), withbovine serumalbumin as astandard.

RESULTS

Although a spectrum of different antigenic determinants is probably present in most type

C viral proteins, three general classes of deter-minants have been described: type-specific

de-terminants, whichdistinguishdifferent isolates from the same type C viral group (e.g.,

Rauscher and Kirsten strains of MuLV);

group-specific determinants shared by a group of viruses isolated from the same species (e.g., MuLV's); andinterspecies determinants shared

by viruses isolated from different species (e.g.,

MuLV's and FeLV's) (la). In general, antisera raised to p30 proteins are directed

primarily

toward group-specific determinants,

although

manyseracontainantibodies of lower titer and

avidity which react with interspecies

determi-nants aswell.

Table 2 shows the results of a series of experiments in which antisera to p30 proteins

weretitrated against radiolabeled p30 proteins purified from the same or different type C viruses. The resultsareexpressedasthe recipro-cal oftheserumdilution requiredtobind20%of the labeled test antigen. An antiserum to the p30 protein of FeLV bound 20% of the 1251_ labeled FeLVtestantigenata serumdilutionof 1:400,000. At a serum dilution of 1:20,000, equivalent binding was obtained with the p30

proteins oftwomurinetypeC viruses(Rauscher

MuLV [Table

21

and the endogenous BALB/c N-tropic virus S2CL3 [not shown]). The cross-reactions between the p30 proteins of FeLV's and MuLV's reflect the presence of the gs-3

determinant (11) and have beenobserved with

many sera prepared from several species of

animals (11, 13, 33, 35, 43, 44). By contrast, equivalentbinding of the p30 proteins ofSSAV, GALV, the endogenous porcine PK(15) virus, the endogenous feline virus RD-114, and the endogenous baboonvirus M7 wasonlyobserved atsignificantly lower dilutionsof theanti-FeLV

p30 serum. A lower-titer antiserum to the p30

protein ofR-MuLV showedareciprocalpattern

ofcross-reaction with the p30 protein of FeLV

and didnot reactstronglywiththep30 proteins

of theothertypeCvirusesstudied.These results

show that theinterspeciesdeterminants shared

by FeLV and MuLV p30 proteins are not as readily detected on the p30 proteins of several

othergroupsofmammalian typeC viruses.

At least twoother major classes of interspe-cies determinants can be identified on type C viral p30 proteins (Table 2). An antiserum to the p30 protein of SSAV reacts strongly with both the SSAV and GALV p30 proteins (cf. 14, 21, 36, 49, 60) andcross-reacts to alesserextent with the p30 proteins of the porcine PK(15)

virusandRauscher MuLV. An antiserum tothe

p30 proteinofthePK(15) virus also cross-reacts with SSAV, GALV, and MuLV p30 proteins.

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TABLE 2. Antigenic cross-reactions betweenp30proteins of type C viruses as measured by immunoprecipitationa

I125-labeledp30 Relative antibody titer

protein Anti-FeLV Anti-R-MuLV Anti-SSAV ] Anti-PK(15) Anti-RD-114 Anti'M7

FeLV 400,000 4,000 400 100 100 400

R-MuLV 20,000 40,000 3,000 3,000 200 300

SSAV 100 200 200,000 5,000 <50 100

GALV 50 200 200,000 10,000 <50 <50

PK(15) 50 < 50 8,000 50,000 < 50 < 50

RD-114 800 <50 400 50 15,000 2,500

M7 1,600 400 400 .50 5,000 8,000

aPurified

p30

proteins were radiolabeled with 1251 and titrated with antisera raised toward homologous and

heterologous p30 proteins. The results are presented as the reciprocal of the serum dilution required for 20%

binding of the labeled test antigen. Results obtained with the homologous antibody-antigen systems are

underlined andreflect the titers of the sera employed.

Neitherserum, however, reacts strongly to the p30 proteins of FeLV, RD-114, or M7. These

results suggest thatthe p30 proteins of SSAV,

GALV, PK(15), and MuLV share an interspe-cies determinant different from that shared by FeLV and MuLV. By contrast, antisera raised tothe p30proteins ofRD-114 andM7show that thep30proteins ofthesetwoviruses arehighly related (cf. 48, 49), whereas neither serum reactsstronglywith thep30proteins oftheother

isolates.

Competition radioimmunoassays were used to further study the interspecies determinants ofp30proteins in an attempt togeneralize these conclusions to other viral isolates. When such

assays are designed with an antiserum and homologous test antigen, the assays are

initi-ated withrelativelyhigh dilutionsofantisera so

that antibodiesbinding group-specific

determi-nants are preferentially selected (cf. Table 2). Cross-reactions

.with

unrelated groups of typeC

viruses arethenrarely detected by competition.

In contrast, competitive radioimmunoassays employing heterologous combinations of anti-sera and labeled test antigens are initiated at lower serum dilutions, permittingthe detection

ofp30 proteinsfromdifferent but relatedgroups ofviruses.

Figure 1Ashows the results of acompetition

assay utilizing an antiserum tothe p30 protein

of FeLV and an iodinated p30 protein from Rauscher MuLV. Disrupted viruses were used

as competing antigens. Both FeLV and

Rauscher MuLV competed efficiently for the labeled test antigen; competition was also

ob-served with the

endogenous

rat viruses RT21C and CCL-38. Similar results have been seen

with endogenous murine type C isolates of various host range types [ATP-124, S2CL3,

S16CL10(I),

R4/B],

with another endogenous

rat virus (VNRK), and with other strains of

FeLV. Only low levels of competition were

observed with SSAV, GALV, PK(15), RD-114, and M7 viruses or with other viruses of these groups(cf. Table 3). These results confirm that

the

p30

proteins ofFeLV and viruses ofrodent origin (rat and mouse) share interspecies

anti-genic determinants that are not readily

de-tectedby these methods inseveral othergroups of type C viruses.

In an assay with an antiserum to the p30 protein of the porcine PK(15) virus and a

radiolabeled SSAV p30 protein (Fig. 1B),

SSAV, GALV, and endogenous porcineviruses

all competed with identical slopes for the la-beled test antigen. All of the mouse viruses

tested cross-reacted inthis assay, as evidenced

by

the reduced

slopes

of the

competition

curves, indicating that MuLV's contain p30 proteins with interspecies determinants

par-tially

related to those found in

SSAV,

GALV,

and endogenous porcine viruses. In contrast,

only limited competition was observed in this

assay with FeLV or with the endogenous rat virusesRT21C, VNRK, and CCL38.

By

replac-ing the labeled SSAV test antigen with a labeled

p30

protein from the endogenous mu-rinevirus S2CL3 (Fig.

1C),

it was againshown that the

p30

proteinsfrom

SSAV,

GALV,

por-cine, and murine viruses share antigenic deter-minants not found in FeLV or

endogenous

rat viruses. Thus, the

p30

proteins of murine vi-ruses contain at least two

distinguishable

classes of interspecies determinants, one set

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SHERR ET AL.

80

6

uZ 60_

4 -J 40

20

0.003 0.01

z

g

n

MICROGRAMS VIRAL PROTEIN

01 10

100

p

z

Lfi J.Lf

n

Ca

80 60

20

UM

FIG. 1. Competitiveradioimmunoassays for p30proteins. Detergent-disruptedtype C viruseswereusedas

sourcesofcompeting antigens.Allassayswereinitiatedwithantiseratoonep30proteinandapurified, labeled p30proteinfromaheterologousvirus. Theamountofcompetingviralprotein requiredtoinitiatedisplacement

of the labeled test antigen reflects both the degree ofpurity of the competing virus preparation and the

sensitivityoftheassaysystem.Intheassaysshown,a20%displacement ofthelabeledtestantigenisobtained

withIto2ngofunlabeled p30proteinidenticaltothetestantigen.Thereagentsemployed in the variousassays were:(A)Anti-FeLVp30: I25I-MuLVp30; (B) Anti-PK(15)p30:I25I-SSA Vp3O; (C) Anti-PK(15)p30:125I-S2CL3

p30; (D) Anti-RD-114 p30:I25I-M7p30. Symbols: MuLV (Rauscher strain); A, S16CL10(I) (endogenous

virusfrom BALBIcmouse); A,AT-124(endogenous virus from NIH/Swissmouse);*, CCL38(endogenousrat

virus); 0,RT21C (endogenous ratvirus); 0,SSAV (infectious primate virus); 0, GALV (infectious primate

virus);V, PK(15) (endogenousporcinevirus); x,FeLV (Rickard

strain);O,

RD-114(endogenous domesticcat

virus);*,M7(endogenous baboon virus).

shared with FeLV's andendogenousrat viruses

(Fig. lA), and a second setshared with SSAV, GALV,andendogenous porcine viruses(Fig. 1B

and C).

By using an antiserum to the p30 protein of

theRD- 114 virus andalabeledp30 proteinfrom

the M7 baboon virus (Fig. 1D), it was shown

that the RD-114 and M7 viruses share

interspe-cies determinantsnotfound in thep30 proteins

of other type C viral groups (49). Identical

re-actions also were seen with other endogenous

feline and baboon type C isolates (CCC and

M28, respectively).Thisassay, then,definesyet

another set of interspecies determinants that

distinguish endogenousbaboon andendogenous

feline type C viruses from the other groups

ofviruses studied.

Table 3 summarizes the resultsobtainedwith

radioimmunoassaysforp30proteins.The slopes of the competition curves generated with

ho-mologous competing antigens are assigned

val-ues of 1.0, and the degree of antigenic

cross-reactionseenwithheterologous competing

anti-gens has been determined by a slope-ratio

method (49). The results generalize the data

showninTable2toadditional viral isolates and

show that at least three distinguishable sets of interspecies antigenic determinants can be

found on p30 proteins of mammalian type C viruses. Goldbergetal. (14a)have alsoreported

similar patterns ofcross-reactivity between the

p30 proteins of these different groups of type C viruses.

To determine whether the cross-reactions

observed between p30 antigens could also be

demonstrated with anothertypeC viralprotein, the immunological properties of the reverse

transcriptasesfromtheseviruseswerealso

stud-ied. Table 4 shows the results of studies in which antisera were used toinhibit the polym-C.

100

a

Z 60

-J 40

20

0.

I I

)- D. _4- 4

n)arrl r) I n If

).003 001

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TYPEC VIRAL ANTIGENS

erases from various viruses. The results are

presented as the relative quantity of immune

IgG required for 30% inhibition of enzyme

activity. Although only data obtained with

respresentative viruses and sera are shown,

quantitatively similar results have been ob-tained with the other isolates from these same

virus groups (Table 1) and with other antisera

preparations.

Table 4 indicates that the antisera to viral polymerases used in thesestudies react prefer-entially with group-specific determinants. Sig-nificantlygreaterquantitiesof immuneIgGare

required to inhibit the activity of heterologous viral enzymes as compared to the amounts

required for comparable levels of inhibition of the homologous polymerases. Like thep30

[image:6.502.54.451.200.391.2]

anti-sera, cross-reactions withheterologous enzymes

TABLE 3. Antigenic cross-reactions betweenp30 proteins of type C viruses as measured bycompetitive

radioimmunoassaya

Cross-reaction

Virus Isolated from: Anti-FeLV: Anti-PK (15): Anti-RD-114:

MuLV SSAV M7

FeLV(Rickard) Domesticcat 1.07 0.12 <0.10

FeLV(Gardner-Arnstein) 1.05 0.11 <0.10

RT21C Rat 0.95 <0.10 0.10

CCL-38 0.95 <0.10 0.10

MuLV (Rauscher) Mouse (1.00) 0.52 0.15

S2CL3 0.97 0.51 <0.10

AT-124 0.98 0.49 0.10

SSAV Woolly monkey 0.21 (1.00) <0.10

GALV Gibbon ape 0.18 1.00 <0.10

PK(15) Pig 0.28 1.02 <0.10

MPK 0.22 1.00 <0.10

RD-114 Domesticcat 0.28 0.11 0.90

CCC 0.23 <0.10 0.98

M7 Baboon 0.27 0.10 (1.00)

M38 0.28 0.11

.99J

aAntigenic cross-reactions were quantitated by comparing the slopes of competition curves. Data were

calculated by obtaining the average slope of at least six separate competition curves for each antigen. The

averageslope of the competition curves for the homologous competing antigen in each assay was assigned a

value of 1.00. The ratio, slope of competition curve (cross-reacting antigen)/slope of competition curve (homologousantigen), isindicated in the table and is an index of the relative degree of cross-reaction between different antigens tested in the same assay (49). The homologous competing antigens are indicated by parentheses.

TAJz4. Inhibition of viralreversetranscriptaseactivity byantiseratoviralpolymerasesa

Viral Relative concentration of immuneIgG required for 30% inhibition of enzyme activity enzyme

tested Anti-FeLV Anti-MuLV Anti-SSAV Anti-GALV Anti-PK (15) Anti-RD-114 Anti-M7

FeLV |(1.0) 32 >50 >50 >15 >50 >50

MuLV l9.2 (1.0) 39 10 5.9 >50 >50

SSAV >50 >50 (1.0) 2.1 3.1 >50 >50

GALV >50 >50 1.8 (1.0) 5.4 >50 >50

PK(15) >50 >50 11 21 (1.0) >50 >50

RD-114 >50 >50 >50 >50 >15 (1.0) 2.0

M7 >50 >50 >50 >50 >15 1.6 (1.0)

aAntiseratopartially purified viral polymerasesofFeLV(Rickard strain), MuLV(Rauscherstrain), SSAV,

GALV,endogenous porcinevirus PK(15),endogenousfeline virusRD-114, and endogenous baboon virus M7

werepreparedandimmuneIgGpurified.Enzyme inhibitioncurves wereobtainedby using serial dilutions of

immune IgG and detergent-disrupted viruses as sources of enzymes. The results have been normalized by

assigningavalue of1.0(indicatedbyparentheses) forthehomologousantibody-antigen systems. The actual

amounts ofimmune IgG required for 30% inhibition of enzyme activity in the homologous systems are:

Anti-FeLV:FeLV, 0.95jg; Anti-MuLV:MuLV, 1.2pg;Anti-SSAV:SSAV, 1.0 ug; Anti-GALV:GALV, 0.81Mg;

Anti-PK(15):PK(15), 3.1 Mg;Anti-RD-114:RD-114, 0.09Mg; Anti-M7:M7, 1.0Mg.

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SHERR ET AL.

canbedetected,and thepatternsobservedwith the reverse transcriptases are similar to those

obtained with p30 antigens (compare Tables 2 and 4).

Antiseratothereversetranscriptasesof FeLV and Rauscher MuLV showed reciprocal

cross-reactivity and did not significantly inhibit en-zymes from other type C viral groups. An

antiserum to the polymerase ofSSAV strongly

inhibited the enzyme from GALV and

cross-reacted to a lesserextent with the polymerases of the porcine PK(15) virus and MuLV. A

similar pattern wasobservedwith anantiserum

tothe reversetranscriptase ofGALV, although a greater degree ofcross-reaction was observed with the murine viral enzyme than with the

PK(15) polymerase. An antiserum to the

PK(15) enzyme strongly inhibited the polymer-ases ofSSAV andGALV and,toalesserextent,

the MuLV reverse transcriptase. Table 4 also shows that the polymerases of the endogenous felinevirus RD-114and theendogenousbaboon virus M7 are closely related antigenically

al-though neither enzyme appears immunologi-callyrelated tothe other reversetranscriptases studied. These data show that three

distin-guishablesets ofinterspecies antigenic

determi-nants can beidentifiedon thepolymerasesfrom

different groups ofmammaliantype C viruses.

DISCUSSION

Cells from a variety of mammalian species contain sequences in their DNA that can code forthe productionofendogenoustype C viruses.

These virogene sequences are thought to be present inboth thesomaticand germ cellsofall

members of the species and are inherited as

integral components ofthecellulargenome (22, 58). Endogenous virogene sequences held in common among related species should be the

direct descendants ofthe same sequence pres-ent in the most recent common ancestor. The

degree of base pair mismatching observed be-tween endogenous virogenes of tworelated spe-cies should then be a relative index ofthetime since the twospeciesdivergedfromoneanother.

For example, sequences related to endogenous

baboon type C viral RNA have been identified

inall the other Old World

monkeys,

thehigher

apes, and man, and the degree ofrelatednessof such sequences to baboon type C viral RNA correlates well with the knowntaxonomic

rela-tionships ofthe primate species (2, 6).

The proteinsofendogenous virusesofrelated species, like proteins encoded byother cellular genes, areexpected to have related amino acid

sequences. Serological studies ofproteins from

different

species

have indicated that

immuno-logical

cross-reactivity

can be

effectively

used

as ameasureof amino acid sequence

dissimilar-ity

and as an index ofthe time of

evolutionary

divergence

ofrelated

species (8,

12,

15, 19, 20,

24,

28, 41).

As

expected,

the

proteins

of

endoge-nous viruses of related

species

show a

high

degree

of

immunological

cross-reactivity.

For

example,

the

homologous proteins

of

endoge-nous rodent

type

C viruses are

antigenically

similar

(11, 13,

14a, 34, 35,

44) as are the

p30

proteins

isolated from several groups of

pri-mates

(47,

50).

However,

in certain cases, the

homologous

proteins

of viruses isolated from

genetically

unrelated

species

ofanimals showan

unexpect-edly

high degree

of

antigenic

relatedness. The

proteins

of

endogenous

viruses isolated from

primates

and domesticcats exhibit an

extraor-dinary degree

of

homology (18,

48, 49, 59)

even

though

these groups of animals have been

evolutionarily

separated

for at;least 80 million years.These results

suggested

that the

RD-114/

CCC group ofdomestic cat viruseswasderived from

endogenous

primate

viruses that infected

theancestorofcertainFelis

species

ata

point

in

recent

evolutionary history,

a conclusion

strongly

supported

by

nucleic acid

hybridiza-tion

experiments (7).

Thus,

animals of one

species

may be infected

by

type

C viruses

derived from a

second,

distantly

related group of

animals, leading

to the

incorporation

ofnew

virogenes

intotheDNA of infected animals

and,

ultimately,

tothe

perpetuation

ofthese genes in

the germ line.

Antigenic

cross-reactions observed between

the

homologous

proteins

of type C viruses iso-lated from unreiso-lated

species

of animals may thus be

explained

by

horizontal transmission of

type

C viruses across

species

barriers. We pro-pose that such events may be more common than

previously thought,

and that

serological

studies may offer the first clues as to the

origins

of certain type C isolates. The

inter-species

determinants shared

by SSAV,

GALV,

and

endogenous porcine

andmurineviruses sug-gest that these viruses

originated

from a

com-mon ancestor. The presence of viruses of the

SSAV-GALV group in

primates,

where

they

are not

endogenous,

would appearto

exemplify

a

relatively

recent infection. Viruses of the

SSAV-GALV groupcontain RNA genomes that are

partially homologous

to sequences in the genomes of murine type C viruses

(5).

That

SSAVand GALVmay bederived from murine viruses that infected

primates

is supported

by

theobservation that sequencesrelated toSSAV

1446 _J. _VIROL.

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(8)

and GALV RNA canbe found in murine DNA

(2) but not in the DNA of normal woolly

monkeys or gibbons (2, 45). Similarly, the

re-lationships observed betweenrodent viruses and viruses of the FeLV group, now horizontally

transmitted in domestic cats, may also reflect

infection ofcats with viruses ofrodent origin. Surveys for related groups of endogenous

virogene sequences in species of animals both

related and unrelated tothose from which type C viruses have been isolated should provide

additional insights into the validity and generality of these interpretations.

ACKNOWLEDGMENTS

We thankLinda Tolerand BarbaraDetwilerforassistance

with these experiments, and Michael M. Lieber for his

suggestions concerningthemanuscript.

Thisworkwassupported by theVirus CancerProgramof

the NationalCancerInstitute.

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