INFECTION AND IMMUNITY, Feb. 1979, p. 472-478 Vol. 23, No. 20019-9567/79/02-0472/07$02.00/0

Specificity of Response to Viral Proteins in Horses Infectedwith Equine Infectious Anemia Virus

HOWARD CHARMAN,1 CEDRIC LONG,`* AND LEROY COGGINS2Viral Oncology Program, Frederick Cancer Research Center, Frederick, Maryland 21701,' and Department

of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 148532

Received for publication 15 November 1978

Three structural proteins of equine infectious anemia virus were purified,labeled with 125I, and utilized in radioimmunoassays with horse sera and antiserato heterologous retroviruses. Whereas radioimmunoassay titers for the majorprotein, p25, were 500- to 1,000-fold higher than titers in immunodiffusion, forclinical purposes these two procedures were equivalent. Antibodies to two low-molecular-weight proteins, p12 and plO, were also found in infected horses, butwith a lower frequency and lower titers. As a rule, only sera positive for p25 alsocontained antibody to p12 and plO. Antisera to the major structural protein ofother retroviruses did not precipitate equine infectious anemia virus p25. Thesesera include antibody to mammalian type C viruses, bovine leukemia virus, visnavirus, mouse mammary tumor virus, squirrel monkey retrovirus, and Mason-Pfizer monkey virus.

We have previously noted many similaritiesbetween equine infectious anemia virus (EIAV)and members of the retrovirus family (2). Thesesimilarities include a virion-associated reversetranscriptase, a high-molecular-weight ribonu-cleic acid genome (2, 8), and a polypeptide com-position more closely resembling non-type Cthan type C viruses. EIAV is horizontally trans-mitted and the genome is not endogenous tohorses (30). No other animal has yet been iden-tified as a reservoir of EIAV (30). The antigenicdeterminants of EIAV p25 do not cross-reactimmunologically with type C or other retrovi-ruses when tested by complement fixation orimmunodiffusion (ID), suggesting that EIAV isnot closely related to other known retroviruses(2). Since distant relationships could be ob-scured by limitations in assay sensitivity, wehave purified EIAV p25, p12, and plO and ex-amined the immunological reactivities of theseproteins by radioimmunoassay (RIA).

MATERIALS AND METHODSVirus. The Wyoming strain of EIAV was grown in

low-passage equine fetal kidney cells according tomethods previously published (21). Virus was isolatedby isopycnic centrifugation, using procedures estab-lished for type C viruses (12).SDS-PAGE. Protein purifications were monitored

by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE), using either 15% cylindrical or5 to 20% linear gradient slab gels and the Laemmlibuffer system (19).Two-dimensional gel electrophoresis. Two-di-

mensional gel electrophoresis, utilizing isoelectric fo-

cusing in the first dimension and SDS-PAGE in thesecond dimension, was performed according to themethods described for both equilibrium (25) andnonequilibrium (27) conditions. A 400-ul amount ofEIAV (110 pg) and 30 1ul of lysis buffer were mixed,and isoelectric focusing was carried out in the firstdimension. The gels were removed and equilibrated inSDS sample buffer, frozen, stored at -70°C, and runin the second dimension.

Isolation of EIAV p25 and pl2. About 10 mg ofpurified virus was solubilized as previously published(1, 31), dialyzed against 0.01 M tris(hydroxy-methyl)aminomethane (Tris)-hydrochloride, pH 7.8,and then passed over a 5-ml diethylaminoethyl-cellu-lose ion-exchange column (Whatmann DE-52, Micro-granular). The unbound proteins were concentrated,and p25 was obtained by Ultragel ACA-44 gel filtrationchromatography. EIAV p12 eluted early in a linear,100-ml, 0 to 0.5 M NaCl gradient.

Isolation of EIAV plO. EIAV was disrupted over-night at room temperature in buffer containing 6 Mguanidine hydrochloride, 20 mM Tris-hydrochloride,pH 7.8, 0.2% /3-mercaptoethanol, and 0.1 mM ethyl-enediaminetetraacetate. Samples were then made 10%in sucrose and applied to a Bio-Gel A 1.5 M column(2.5 by 80 cm) equilibrated with the above buffer.Fractions of 1.5 ml were collected at a flow rate of 10ml/h and assayed for single-stranded calf thymus de-oxyribonucleic acid (DNA)-binding activity (20). TheDNA-binding peak was found, and fractions werepooled in and surrounding the peak. The pools weredialyzed against 0.1 M ammonium bicarbonate, lyo-philized to dryness, and analyzed by SDS-PAGE.Fractions within the DNA-binding peak containedspecies of a major- and a minor-molecular-weightform. Fractions pooled outside the DNA-binding peakcontained only the minor low-molecular-weight spe-cies. The major protein represented in the DNA-bind-

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IMMUNE RESPONSES TO EIAV PROTEINS 473

ing peak corresponded to the basic plO protein isolatedfrom murine viruses (20). The low-molecular-weightprotein used in the present study was shown by isoe-lectric focusing to be an acidic protein. To distinguishthese p10 subspecies, they will be referred to as plOa(acidic) and plOb (basic).

Antisera. Antisera from horses naturally or exper-imentally infected with EIAV were characterized forantibodies to EIAV p25 by ID according to publishedmethods (10). Some of these sera were available froma previous study of an epizootic of EIAV (33). Consul-tation sera from suspected cases of EIA were serareferred to one of the authors (L.C.) for confirmationof the presence or absence of EIAV p25 antibodies.Antisera recognizing mammalian type C virus inter-species p30 determinants were obtained by sequentialimmunization with several type C virus p30's (11).Sera produced in this manner reacted with all type Cviruses available in this laboratory in ID and comple-ment fixation. Goat antisera to bovine leukemia virus,feline leukemia virus (FeLV), woolly leukemia virus,and gibbon ape leukemia virus, as well as high-titeredpig anti-goat immunoglobulin G, were obtainedthrough Roger Wilsnak (Huntingdon Research Labo-ratories, Brooklandville, Md.). Similar antisera wereproduced at the Frederick Cancer Research Center.Ashley T. Haase (San Francisco Veterans Administra-tion Hospital) kindly supplied a goat antiserum tovisna virus p27. Charles Benton (Viral Oncology Pro-gram, Frederick Cancer Research Center) supplied agoat antiserum to murine mammary tumor virus p27.A goat antiserum to squirrel monkey retrovirus wasthe gift of Larry Arthur (Frederick Cancer ResearchCenter). A goat antiserum to lethally irradiated EIAV(14, 15) was produced at the Frederick Cancer Re-search Center, as was a serum from a goat immunizedwith Mason-Pfizer monkey virus p25.

RIAs. Purified proteins were iodinated either byusing the chloramine T procedure (13) or with iodogen(6). Antibody titrations were performed by using adouble-antibody procedure (1) with 300 tig (1.7 mM)of phenylmethanesulfonyl fluoride per ml in all buffers(7).

RESULTSPurity ofEIAV proteins. The complexity of

whole EIAV is evident in the two-dimensionalelectrophoresis pattern shown in Fig. 1. Threemajor protein species based on molecular weightare evident from this analysis. Within each spe-cies there were multiple forms that differed incharge and only slightly in molecular weight.One species migrated between trasylol (molecu-lar weight, 6,000) and cytochrome c (molecularweight, 12,200); based on a comparison to pro-teins from Rauscher leukemia virus, we desig-nated this plO. Subspecies were present in thismolecular-weight region *hich differed mark-edly in isoeletric points. In the present study,the acidic component plOa was obtained in ho-mogeneous form as shown by two-dimensionalelectrophoresis (Fig. lb) and used in the immu-nological studies. The major basic component,

plOb (seen in the right of Fig. la), most probablycorresponded to the DNA-binding protein foundin murine type C viruses. Another species mi-grates between cytochrome c and ribonuclease(molecular weight, 13,800), which is the p12 re-gion of Rauscher leukemia virus. The third spe-cies was the major virion protein, p25, whichmigrated slightly less than the light chain ofgamma globulin (molecular weight, 30,000). Thesubspecies present in the p12 and p25 regionsdid not differ greatly in isoelectric points, andfurther separation was not attempted.The purity of the proteins after iodination and

used in the RIA to be described is shown in Fig.2. After SDS-PAGE, each protein migrated as asingle molecular species of greater than 95%purity.RIA of EIAV p25, p12, and plO. Table 1

shows that only the antiserum from a goat spe-cifically immunized with EIAV contained pre-cipitating antibodies to EIAV proteins. Refer-ence antisera from goats immunized with visnavirus p27, Mason-Pfizer monkey virus p25, mu-rine mammary tumor virus p27, and bovine leu-kemia virus lacked antibodies to mammaliantype C viruses, including RD-114, baboon leu-kemia virus, FeLV, and simian sarcoma virus,and did not bind EIAV protein. Most impor-tantly, two broadly reacting antisera that precip-itate all type C viruses available to us did notprecipitate EIAV p25.Detection of EIAV p25 antibodies in

horse sera using RIA. We found that RIAtiters were roughly 500 to 1,000 greater than IDtiters at limits of detection. However, this madelittle difference in detecting clinically infectedanimals. We found that, with only one exception,ID-positive sera had high-titered antibodies toEIAV p25 (Table 2). Conversely, there was avery low incidence of falsely negative ID tests.There were a total of two consultation sera andfour epizootic sera that contained EIAV p25antibodies by RIA but were ID negative (Table2). Two of these sera had marginal titers (1:40and 1:80) and the others were low: 1:160, 1:160,1:320, and 1:1,280.Demonstration that p12 and plO are tar-

gets of the immune response to EIAV. Itwas previously established by less sensitivemethods (10) and RIA (9) that EIAV p25 wasthe major target of the host immune response toEIAV infections. An additional reactivity wasdetected in other sera directed against an anti-gen of about 15,000 daltons, presumably theprotein we designated p12 (32). To further char-acterize the response to EIAV infections, weanalyzed sera that were already characterizedfor p25 antibodies for the presence or absence ofantibodies to EIAV p12 and plO. p12 and plO

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474 CHARMAN, LONG, AND COGGINS

Ac d ka

Basic.b

'*'chain bl~

l..o i.'i.* gha t

_ h a r

-globi.P: -1'

-.. Thbonuc.e-ase-< Cvtochrcr'!e K

.t ,,.. -s.2!-

[Sv

FIG. 1. (a) Two-dimensional gel electrophoresispattern ofwhole EIA Vrun according to the nonequilibriumpH gradient electrophoresis technique of O'Farrell et al. (27) as described in the text. Molecular weights ofproteins were estimated by comparing mobilities of Trasylol (6,000), cytochrome c (12,200), ribonuclease(13,800), and light and heavy chains ofgamma globulin (30,000 and 50,000). Notations to the left of the figureshow the molecular weights corresponding to plO, p12, and p25. (b) Two-dimensional electrophoresis patternofpurified plOa obtained from guanidine-agarose chromatography as described in the text.

antibodies were detected in about half of thesesera (Table 3). Although all the proteins were

comparably immune precipitable by the hyper-immune goat serum, the titers of p12 and plOantibodies were much lower than titers to EIAVp25. Thus, the geometric mean titer of 40 serawith p25 antibodies was 1:4,430, that of 24 serawith p12 antibodies was 1:95, and that of 20 serawith plO antibodies was 1:70. Several of thesesera were used to establish RIAs that detectedthe appropriate antigen in isoelectrically focusedEIAV or on EIAV fractionated on agarose col-umns in the presence of 6 M guanidine hydro-chloride.With one exception, only p12 and plO antibod-

ies were found in sera also containing p25 anti-bodies. The exception occurred in an experimen-tally infected horse that developed EIAV p25antibodies a few days later. Clearly, EIAV p25is the major target of the host immune response.

DISCUSSIONRIAs have proven to be useful tools for ser-

oepidemiological investigation of horizontallypassaged retroviruses. Thus, in previous studies,we found antibodies to murine leukemia virusp30 in the sera of human cancer patients im-munized with Rauscher murine leukemia virusand gibbon ape leukemia virus p30 antibodies in

Acidic

Plo ---

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IMMUNE RESPONSES TO EIAV PROTEINS 475

1.0

0.8

0.6

0.4

0.2

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ZD 0.8

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°.- 0.6

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1.0

0.8

0.6

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29 18.5 14.3

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0.2 0.4 0.6 0.8 1.Relative Mobility

FIG. 2. Five microliters ofca. 106 to 107 c,labeled EIAV proteins were mixed with 2mixture containing 5 ug each of standard p~indicated molecular weights (MW): bovinebumin, 68,000; carbonic anhydrase, 29,000;toglobulin, 18,500; and lysozyme, 14,300. Thwere diluted with an equal volume of 2xsample buffer (19) for a final sample buffer co

tion of 0.0625 M Tris-hydrochloride, pH 7.8,10% glycerol, 5% 2-mercaptoethanol, andbromophenol blue as a marker dye. Theprotheated for 5 min in a boiling-water bath, el

healthy contacts of leukemic gibbons (5). In5.0 addition, FeLV p30 antibodies were demon-

strated in the sera of healthy cats from high-incidence leukemia environments, but not in the

4.5 sera of healthy normal cats or in the sera of catsviremic with FeLV and suffering from a varietyof neoplastic or non-neoplastic diseases (3, 4).

4.0 Although antiviral antibodies were demonstra-

TABLE 1. Lack of reactivity of reference goatantisera to various retroviruses with EIAVproteins

RIA titera vs '25I-labeled EIAVSerum protein

p25 p12 plOEIAV 64,000 10,240 10,240Interspecies p30 <20 <20 <20Mammalian type C vi- <20 <20 <20

rusesbMPMV (several sera)c <20 <20 <20SMRVd <20 <20 <20MuMTV p27' <20 <20 <20Visna p27 <20 <20 <20

4.0 _ a Reciprocal of serum dilution for 10 to 20% binding.o All reactants were diluted in RIA buffer, which con-*> sisted of 0.05 M Tris-hydrochloride, pH 7.8, containing

0.15M NaCl, 0.5% bovine serum albumin, 0.1% sodiumazide, 0.4% Triton X-100, and 1.7 mM (300 tg/ml)phenylmethanesulfonyl fluoride. For antibody titra-tions, 50 pi of '25I-labeled protein, 50 ,l of antibody,and 100 ul of RIA buffer were incubated for 1 h at37°C and overnight at 4°C, at which time an excess of

5.0 pig anti-goat immunoglobulin G serum and 0.4 ml ofrinse buffer (0.01 M Tris-hydrochloride, pH 7.8, 0.1%Triton X-100, 0.1 M NaCl, and 0.001 M ethylenedia-minetetraacetate) were added. Tubes were incubated

4.5 for an additional 1 h at 37°C and 4 h at 4°C. Aftercentrifuging for 15 min at 2,500 rpm in a Sorvall RC2B centrifuge, using HS4 rotors, supernatant fluidswere aspirated to waste, and precipitate radioactivity

4.0 was determined in a Searle model 1285 autogammacounter. Results are expressed as the serum dilutiongiving 10 to 20% binding after subtracting non-specif-ically bound counts.

b Including sera to RD-114, FeLV, simian sarcomavirus-1, and gibbon ape leukemia virus; all these anti-sera and others gave titers >10,000 versus homologous

O antigens.'MPMV, Mason-Pfizer monkey virus.d SMRV, Squirrel monkey retrovirus.eMuMTV, Murine mammary tumor virus.

pm Of 1zoi.0,ul of aroteins ofserum al-beta-lac-eproteinsLaemmlioncentra-t,2% SDS,0.001 Mtems werefectropho-

resed at 1 mA/gel for 18 h, stained with Coomassiebrilliant blue, and destained, and the position ofmarker proteins was noted. The fractions were cutinto 1-mm sections with a razor blade device, and theradioactivity ofeach fraction was then determined inan autogamma counter. The radioactivities wereplotted as a function of mobility relative to the low-molecular-weight tracking dye and known molecular-weight markerproteins. (A) EIA Vp25; (B) EIA VpI2;(C) EIAVplO.

5.0

4.5

68 29 18.514.3

-C

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II

68 29 18.5 14.3

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476 CHARMAN, LONG, AND COGGINS

TABLE 2. Analysis of equine sera examined forEIAVp25 antibodies by RIA

No. posi- Ge triSera tive/no. mean titer Range

tested ma ie

Consultation seraID positive 61/62a 6,200 320-40,960ID negative 2/8 113 40-320

Epizootic seraID positive 13/13 3,200 640-40,960ID negative 4/205b 225 40-1,280a One ID-positive serum did not bind EIAV p25.

Whether this represents a false positive ID result oran immune response to some other viral antigen is notknown.

b Titers as follows: 80, 160, 160, and 1,280.

TABLE 3. Presence of antibodies to EIAVp12 andp1O in experimentally or naturally infected horsesera selected for the presence or absence ofEIAV

p25 antibodiesNo. positive'/no.

Protein tested vs protein Geometric Rangemean titer RagNegative Positive'

p25 0/29 40/40 4,430 160-40,960p12 1C/29 24/40 95 40-1,280plo 0/29 20/40 75 20-320

a Positive results reflect >10 to 20% binding at a 1:20 or higher serum dilution. In this assay a controlgoat serum had titers to EIAV p25, p12, and plO,respectively, of 1:64,000, 1:10,240, and 1:10,240.

b Anti-p25 status.¢ This serum had a titer of 1:40 and was from a horse

experimentally inoculated with EIAV about 2 weekspreviously. The following day the antibody titers toEIAV p25, p12, and plO were 1:160, 1:40, and 1:40,respectively, whereas 3 days later the respective titerswere 1:2,560, 1:640, and 1:160.

ble by RIA techniques, they were not detectableby ID.The present study extends our knowledge of

the immune response to EIAV, another retrovi-rus. For some years, infected horses were knownto form high-titered precipitating antibodies toa viral protein, later found to be a 25,000-daltonpolypeptide (10). These antibodies are demon-strable by ID and serve as the basis for theCoggins test, which has been used in many statesto certify horses as free of EIAV. There has beensome concern regarding the specificity of the IDtest, even though it has been possible to mark-edly reduce the incidence of EIA by destroyingor isolating positive reactors. Comparison of IDand RIA results clearly demonstrates the valid-ity of the ID test for detecting EIAV infections.Thus, with a single exception in over 60 sera,high-titered EIAV p25 antibodies were demon-strated in ID-positive sera. Conversely, only a

few positive responses were detected by RIA insera that were ID negative. This is an importantresult in that: (i) it argues that the simpler IDtest is adequate to monitor EIA infection inhorses; and (ii) it sheds some light on the path-ogenesis of this disease.A similar situation pertains to bovine leuke-

mia virus infections (22, 23). EIAV is transmittedby insect vectors or by blood-contaminatedfomites. Experimentally, transmission is easilyaccomplished by small amounts of blood con-taining infected macrophages. Like bovine leu-kemia virus and visna virus infections, and inmarked contrast to FeLV, free extracellular virusis difficult to demonstrate. Also in contrast toFeLV but similar to visna virus and bovine leu-kemia virus, virtually every horse with EIA hashigh-titered antibodies to p25. The importantquestion, then, is why EIAV persists in the faceof the ability to form high-titered antibodies toEIAV proteins. The many immunopathologicalfeatures of EIA (hemolytic anemia, at least par-tially immune complex mediated; subclinical im-mune complex glomerulonephritis, Aschoff bod-ies in myocardial tissue) raise the possibility thatthe immune response may in some way contrib-ute to the development of disease. Alternately,envelope antigenicities of infecting viruses may"drift" during the course of disease (18). A sim-ilar phenomenon has been shown to occur invisna virus (24).Toma (32) has previously reported antibodies

to a second viral protein of about 15,000 daltons.Ishizaki et al. (17) have also reported antibodiesto p15 in horse sera. We have been able to detectantibodies to both p12 and p10 proteins of EIAV.These responses occurred in horses with p25antibodies, were less prevalent, and had a muchlower titer. Clearly, p25 was the major target ofthe immune response.These sensitive RIAs have not permitted de-

tection of immunological cross-reactions be-tween EIAV and other retroviruses. This is inspite of our recent ability to demonstrate that ahorizontally passaged avian virus, reticuloendo-theliosis virus, is immunologically related tomammalian type C viruses (H. Charman, R.Gilden, and S. Oroszlan, J. Virol., in press), aresult substantiated by primary amino acid se-quence data (16). Thus, whether EIAV is re-stricted to the Equidae or present as an endog-enous virus in some other species remains amoot point. Although the importance of verticaltransmission of retroviruses has received consid-erable emphasis in the past, horizontal transmis-sion clearly deserves equal emphasis (32).The resolution observed in the two-dimen-

sional gel electrophoresis pattern ofwhole EIAVunderscores the complexity involved in inter-

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IMMUNE RESPONSES TO EIAV PROTEINS 477

preting immunological studies with purified viralproteins. In the present study, microheterogene-ity was found in virtually every major virionprotein. Charge microheterogeneity in murinetype C virus p30 has been observed previously(28). There are several possible explanations forthis phenomenon. The possibility of cell or se-

rum contamination seems remote since the pu-

rified proteins used in this study reacted in im-munoassays with antibodies present in horsesnaturally infected with EIAV. Others have ob-served that two or more spots on a two-dimen-sional gel may be the product of a single gene.

The major capsid protein of simian virus 40consists, for example, of a major protein spotand minor species varying in degree of phospho-rylation (26). Furthermore, translation errors

can also occur when eucaryocytic or procaryoticcells are stressed by amino acid starvation. Thefaulty proteins resolve into a series of spots withmolecular weights similar to that of the originalprotein but differing in isoelectric points (29).There is, therefore, a basis for translational or

post-translational alterations which could ac-

count for the complexity observed in EIAV.

ACKNOWLEDGMENTS

This work was supported by the Virus Cancer Program,contract N01-CO-75380, National Cancer Institute.We acknowledge the expert technical assistance of Sharon

Bladen, Richard Snead, and Martin White and the continuedinterest and support of Raymond Gilden.

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2. Charman, H. P., S. Bladen, R. V. Gilden, and L.Coggins. 1976. Equine infectious anemia virus: evi-dence favoring classification as a retrovirus. J. Virol.19:1073-1079.

3. Charman, H. P., M. B. Gardner, R. M. McAllister, N.Kim, and R. V. Gilden. 1976. Humoral immune re-

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4. Charman, H. P., N. Kim, R. V. Gilden, W. D. Hardy,Jr., and M. Essex. 1976. Humoral immune responses

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5. Charman, H. P., N. Kim, M. White, H. Marquardt, R.V. Gilden, and T. Kawakami. 1975. Natural andexperimentally induced antibodies to defined mamma-lian type C virus proteins in primates. J. Natl. CancerInst. 55:1419-1424.

6. Charman, H. P., H. Marquardt, R. V. Gilden, and S.Oroszlan. 1977. Immunologic characterization of gp7Oand gp45 from Rauscher murine leukemia virus. Virol-ogy 83:163-170.

7. Charman, H. P., M. H. White, R. Rahman, and R. V.Gilden. 1976. Species and interspecies radioimmunoas-says for rat type C virus p30: interviral comparisons andassay of human tumor extracts. J. Virol. 17:51-59.

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homology between reticuloendotheliosis virus p30 andp3Os of mammalian retroviruses. Proc. Natl. Acad. Sci.U.S.A. 75:2708-2712.

17. Ishizaki, R., R. W. Green, and D. P. Bolognesi. Thestructural polypeptides of equine infectious anemia vi-rus. Intervirology 9:286-294.

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20. Long, C. W., R. Berzinski, and R. V. Gilden. 1977.Immunologic studies of the low molecular weight DNAbinding protein of murine oncornaviruses. Int. J. Cancer19:843-850.

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23. Miller, J. M., and M. J. Van Der Maaten. 1974. Acomplement-fixation test for the bovine leukemia (C-type) virus. J. Natl. Cancer Inst. 52:1699-1702.

24. Narayan, O., D. Griffin, and J. Chase. 1977. Antigenicshift of Visna virus in persistently infected sheep.Science 197:376-378.

25. O'Farrell, P. H. 1975. High resolution two-dimensionalelectrophoresis of proteins. J. Biol. Chem. 250:4007-4021.

26. O'Farrell, P. Z., and H. M. Goodman. 1976. Resolutionof simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the majorcapsid protein. Cell 9:289-298.

27. O'Farrell, P. Z., H. M. Goodman, and P. H. O'Farrell.1977. High resolution two-dimensional electrophoresisof basic as well as acidic proteins. Cell 12:1133-1142.

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Gilden. 1974. Murine type-C virus group-specific anti-gens: interstrain immunochemical, biophysical andamino acid sequence differences. J. Virol. 14:1559-1574.

29. Parker, J., J. W. Pollard, J. D. Friesen, and C. P.Stanners. 1978. Stuttering: high-level mistranslation inanimal and bacterial cells. Proc. Natl. Acad. Sci. U.S.A.75:1091-1095.

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