Vol. 19, No. 2

JOURNAL OF VIROLOGY, Sept. 1976, p. 1073-1079 Copyright (C 1976 American Society for Microbiology

Printed in U.S.A.

Equine Infectious Anemia Virus: Evidence Favoring Classification as a Retravirus HOWARD P. CHARMAN,'* SHARON BLADEN,' RAYMOND V. GILDEN,' Flow Laboratories, Inc., Rockville, Maryland 20852

AND

LEROY COGGINS2

Received for publication 23 March 1976

Equine infectious anemia virus (EIAV) has a density of 1.154 g/cm3 in sucrose, high-molecular-weight RNA similar in size to Rauscher murine leukemia virus, and an internal virion reverse transcriptase that utilizes the synthetic RNA template poly(rA) but not the synthetic DNA template poly(dA), both with (dT),2 as primer. Although capable of utilizing manganese at low concentrations (approximately 0.1 mM), EIAV reverse transcriptase showed highest activity in the presence of 9 mM magnesium. The major protein of EIAV has a slightly lower molecular weight than the comparable protein of type C viruses and coelectrophoresed with '251-labeled p25 of Mason-Pfizer monkey virus. A reference horse serum with antibodies to the major EIAV protein reacted only with EIAV and not with other type C or non-type C retraviruses. Reciprocally, a broadly reactive serum to type C virus p30s and specific sera to a variety of non-type C retraviruses did not react with EIAV. We recommend the inclusion of EIAV in the family Retraviridae. a

Equine infectious anemia (EIA) is characterized by fever, complement- and non-complement-dependent hemolytic anemia, bone marrow depression, and subclinical immune-complex glomerulonephritis. Historically one of the first diseases shown to be due to a virus, EIA is usually transmitted by insect vectors (35) but may also be spread by fomites contaminated with infected horse blood (31, 32). Mare-to-foal transmission has been documented (20). The clinical spectrum of EIA is variable: most infected horses become asymptomatic carriers, others may have a chronic disease characterized by exacerbations and remissions, and a third group has a rapidly progressive fatal course. Once infected, horses remain viremic for life. Whereas the clinical and epidemiological aspects of EIA have been exhaustively characterized and recently reviewed (21), knowledge of the molecular biology of EIA virus (EIAV) has been retarded due to the lack of a tissue culture system for obtaining virus in the quantities required for characterization. The recent development of such a system (22) now

reverse transcriptase, and a polypeptide composition similar to that of certain other retraviruses. Based on these findings, EIAV should be included in the family Retraviridae (6).

MATERIALS AND METHODS EIAV isolation. Tissue culture fluids of an equine fetal kidney cell line (EFK-2) chronically infected with EIAV, Wyoming strain, were clarified by lowspeed centrifugation and precipitated with polyethylene glycol 6000 as previously published (22) or purified by sedimentation in sucrose density gradients and concentrated by pelleting (11). Other retraviruses. The following type C and nontype C viruses used in this study were prepared by conventional methods (29): rat leukemia virus, feline leukemia virus (FeLV), hamster leukemia virus, Rauscher murine leukemia virus (R-MuLV), RD-114 virus, woolly monkey leukemia virus (WoLV), gibbon ape leukemia virus (GaLV), baboon leukemia virus, Mason-Pfizer monkey virus (MPMV), murine mammary tumor virus (MMTV), and bovine leukemia virus (BLV). Purification of MPMV p25. The major structural protein of MPMV was isolated by chromatography on G-100 Sephadex. After labeling with 125I using permits the biochemical and immunological the chloramine T procedure (12), the protein was rechromatographed on G-100 Sephadex columns. analysis of EIAV. We show herein that EIAV has a high-molec- The antigen prepared in this manner consisted of a zone of radioactivity in sodium ular-weight RNA genome, a virion-associated single symmetrical dodecyl sulfate (SDS)-polyacrylamide gel electroof I Present address: Frederick Cancer Research Center, phoresis with an apparent molecular weight 25,000. MD 21701. Frederick, 2 Present address: Research Characterization of the viral genome. Monolayer Laboratory for Equine Infectious Diseases, New York State Veterinary College, Cornell cultures of EFK-2 cells were grown at 37°C for 18 h in 32-ounce (0.95-liter) bottles containing 20 ml of University, Ithaca, NY 14850. 1073

1074

CHARMAN ET AL.

media and 1 mCi of [PH]uridine. After an initial medium harvest, fresh, unlabeled medium was added and the cultures were incubated for an additional 24 h. The combined sample was centrifuged at 3,000 rpm for 10 min to remove cellular debris and then centrifuged at 27,000 rpm for 90 min in a no. 35 rotor (Beckman Instruments, Inc., Fullerton, Calif.). The pelleted virus was resuspended in 2 ml of TNE buffer containing 0.01 M Tris-hydrochloride, pH 7.4, 0.1 M sodium chloride, 0.005 M EDTA and banded in a linear 20 to 60% (wt/vol) sucrose density gradient at 35,000 rpm for 2.5 h in an SW-41 rotor. Fractions in the 1.15- to 1.16-g/cm: density range were pooled, diluted with TNE to less than 10% sucrose, and then pelleted at 35,000 rpm for 90 min in an SW41 rotor. The pelleted virus was resuspended in 2 ml of TNE. SDS was added to a final concentration of 1%, and the virus was overlayed on a 12-ml 15 to 30% (wt/vol) linear sucrose density gradient in TNE containing 0.01% SDS and centrifuged at 35,000 rpm for 150 min at 10°C. Fractions (-0.8 ml) were collected by bottom puncture, and the radioactivity of samples was determined in a liquid scintillation counter (Beckman Instruments, Inc.). This procedure was as previously published (27). Characterization of EIAV reverse transcriptase. (i) Manganese assays. Reaction mixtures of 50 ,l consisted of: viral protein, 640 Ag/ml; Triton X-100, 0.1%; poly(rA)-oligo(dT), 5 gtg/ml, or poly(dA)oligo(dT), 50 ,ig/ml; 0.05 M glycine buffer, pH 8.3; 0.15 M NaCl; 0.02 M dithiothreitol; and 0.0001 M

MnCl. (ii) Magnesium assays. Magnesium assays were performed with the above concentrations of virus, detergent, and template-primer but differed in that 0.05 M Tris-hydrochloride, pH 8.3, 0.04 M NaCl, and 0.005 M dithiothreitol were used and MgCl2 was used at 0.009 M. [:3H]TTP was used at a final concentration of 2 mM. Reactions were incubated for 30 min at 30°C, precipitated with 5% trichloroacetic acid, filtered on membrane filters (Millipore Corp., Bedford, Mass.), dried, and counted in a liquid scintillation counter. (iii) Determination of divalent cation requirements. Incubation mixtures were as above, except that divalent cation concentrations were varied from 0 to 0.080 M (11, 15, 23, 30). Analysis of viral polypeptides. Concentrated virus was analyzed by SDS-polyacrylamide gel electrophoresis using commercially available gels (Biophor; Bio-Rad Laboratories, Richmond, Calif.) and the phosphate buffer system of Weber and Osborne (37). Molecular weight markers in a companion gel from the same run included: ovalbumin, 43,000; carbonic anhydrase, 29,000; lysozyme, 14,300. Additional gels run as controls included FeLV, BLV, BLV p24, and '2I-labeled MPMV p25. The latter was subjected to co-electrophoresis with EIAV. Gels were subjected to electrophoresis for 7.5 h at 8 mA/ gel, cut at the middle of the tracking dye, fixed, stained, and electrophoretically destained. The position of protein bands of radioactive gels was noted during slicing, and the radioactivity of individual

J. VIROL.

slides was determined in a gamma counter (Nuclear-Chicago Corp., Des Plaines, Ill.). CF tests. Complement fixation (CF) tests were performed as previously published (29), using 4 units of antibody of a reference antiserum derived from an infected horse (4, 26). This serum reacted in CF only with cells shedding EIAV and not with normal cells from several species. Antisera. Sera were obtained from guinea pigs immunized with electrofocused-purified p3Os of FeLV, WoLV, and baboon leukemia virus. Sera reactive with the p30s of MuLV and the p25 of MPMV were obtained by immunizing guinea pigs with purified, detergent-disrupted virus. A broadly interspecies-reactive goat serum was obtained by sequential immunization with FeLV, RD-114, GaLV, and WoLV p30s. This serum reacts with all existing type C viruses in CF and binds all '25I-labeled mammalian p30s tested at high serum dilutions (50% binding titers of 50,000 to 100,000, with 10% of titers roughly fivefold higher (250 to 500,000). Radioimmunoprecipitation assays. Radioimmunoassays using highly purified viral p30s and 39% final concentration of ammonium sulfate to separate antigen-antibody complexes were performed as previously reported (1-3).

RESULTS Characterization of EIAV genome. [3H]uridine-labeled EIAV banded in sucrose at a density of 1.154 g/cm3. R-MuLV analyzed under similar conditions banded at 1.166 g/cm3. Fractions around the peak of each gradient were pooled, diluted, pelleted, and incubated in the presence of SDS. When centrifuged through 15 to 30% (wt/vol) linear sucrose density gradients in the presence of 1% SDS, EIAV RNA sedimented at a rate closely approximating that of labeled R-MuLV RNA (Fig. 1), i.e., -70S. Characterization of EIAV reverse transcriptase. Disrupted EIAV polymerized [3HTITP in the presence of the synthetic template-primer poly(rA) oligo(dT). Enzyme activity was linear with respect to protein concentration and with time for up to 90 min. No activity was found with poly(dA) * oligo(dT), a template generally considered specific for cellular DNA polymerases. DNA synthesis occurred only in the presence of detergent, indicating that the enzyme was inside the virion. Endogenous polymerase activity was not demonstrated under the conditions used. The reactivities of EIAV reverse transcriptase in the presence of the synthetic polymers using optimal manganese (0.1 mM) and magnesium (9 mM) concentrations are shown in Fig. 2A and B, respectively. Divalent cation requirements. Figure 3 shows that although EIAV reverse transcriptase utilized manganese at low cation concen-

EQUINE INFECTIOUS ANEMIA VIRUS

VOL. 19, 1976

6.0 cs0

x 4.8

I 4.

._

R-MuLV

3b

RNALV

2.4

I

1.2j , 10

5

| 15

Fraction Number FIG. 1. Charac terization of EIAV RNA. EIAV RNA was isolatedI by sedimentation in a linear 15 to 30% (wtlvol) sucr(ose density gradient in the presence of SDS as detaile d in Materials and Methods. The position of R-MuLV RNA isolated under the same conditions is indicated. 0

16 --A

Mn-o.1mM

B

Mg+ *9mM

x~ 14 1210

_

2

0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (Minutes) FIG. 2. Characterization of EIAV reverse transcriptase. Polymerase reactions were performed as described in Materials and Methods using 0.1 mM Mn2+ (A) or 9 mM Mg2+ (B) final concentration. In both panels U represents the synthetic RNA templateprimer poly(rA) * oligo(dT) and * represents the synthetic DNA template-primer poly(dA) * oligo(dT). CO)

trations (0. 1 mM), the enzyme was clearly most active with magnesium with an optimal concentration of about 9 mM. Analysis of the protein composition of EIAV. Disrupted EIAV resolved into multiple bands in 12% SDS-polyacrylamide gels, with a major protein zone having an apparent molecular weight of about 25,000. This protein coelectrophoresed with 125I-labeled MPMV p25

1075

and was clearly smaller than the major protein of FeLV, which has a generally accepted molecular weight of 27,000. The polypeptide composition of FeLV, EIAV, and BLV is included in Fig. 4 for comparison. Although the MPMV protein is generally referred to as p27 (38), our recent and continued experience is that this protein electrophoreses with a lower apparent molecular weight. Serological reactivities of EIAV. A reference horse serum reacting with the major structural protein of EIAV did not precipitate ''Ilabeled FeLV, baboon leukemia virus, or WoLV p30s. In addition, this serum did not precipitate '2>I-labeled MPMV p25. These results are summarized in Table 1. CF reactions of EIAV antisera and antisera to several type C and non-type C viruses are shown in Table 2. Note that a broadly reacting antiserum made by sequential immunization of a goat with purified p30s reacted with all type C viruses, but not with purified EIAV, under conditions of antigen sufficiency. The antiserum to EIAV reacted only with EIAV and not with any other type C or non-type C virus. DISCUSSION EIAV has for some time been thought to be related to RNA tumor viruses. This was initially based on its morphological resemblance to myxoviruses (19, 25, 33). The presence of an RNA genome and an internal group-specific antigen was further circumstantial evidence (4, 21, 24, 26). The current study clearly demonstrated that EIAV has a high-molecular-weight RNA genome and virion-associated reverse transcriptase and should therefore be included ci 0 U

16. 0

0.4 C,

0

0025OO5W0Q2OQ4

2 4 6 8 10 20 40 80 Final Cation Concentration (mM)

FIG. 3. Divalent cation requirements of EIA V polymerase. Polymerase reactions were performed as described in Materials and Methods and the legend to Fig. 2, except that divalent cation concentrations were varied between 0 and 80 mM. Symbols: *, Mn2+; EO, Mg2+.

1076

CHARMAN ET AL.J.Vo. J. VIROL.

27V. 24j 21-

C

_ h4.5

0

CD0

0

CN4

0-~~~~~~~4.0

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~~~~~~EIAV

a. 12

-

9

LV ~~~~~~~B FLV

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~g.it~

6-

0

0.2

0.4

0.6

0.8

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Relative Mobility FIG. 4. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of EIAV, BLV, and FeLV. SDS-PAGE performed as described in Materials and Methods using 12% gels available from a commercial source. EIAV (100 1.d =70 M.g of virus protein) and 25 p.l of '251-labeled MPMV p25 (88,000 cpm = 4 ng) were subjected to co-electrophoresis for 7.5 h at 8 mA/gel, stained with Coomassie brilliant blue, and destained, and the position of stained bands was carefully noted. The gel was sliced with a razor blade device into 1 -mm sections, and the radioactivity was determined in a gamma counter and plotted as a function of mobility relative to the low-molecular-weight tracking dye bromophenol blue. Other controls in the same gel run were BLV (100 pl1 = 80 pg) and molecular-weight markers (X) as follows: ovalbu min, 43,000; carbonic anhydrase, was

29,000; lysozyme, 14,300.

TABLE 1. Radioimmunoprecipitation reactions of '25I-labeled type C and non-type C virus proteins with a reference serum from a horse with EIA Antibody titers" against 1251-labeled: Serum

MPMV p25

Horse anti-EIA GP-MPMVb

FeLV p27