during Parallel Persistent Infectionst

Transcription

1 JOURNL OF VIROLOGY, pr. 1987, p X/87/ $02.00/0 Copyright 1987, mericn Society for Microbiology Vol. 61, No. 4 Course nd Extent of Vrition of Equine Infectious nemi Virus during Prllel Persistent Infectionst SUSN L. PYNE,' OLIVI SLINOVICH,'t SUZNNE M. NUMN,' CHRLES J. ISSEL,2'3 ND RONLD C. MONTELRO1* Deprtments of Biochemistry' nd Veterinry Science,2 nd Louisin griculturl Experiment Sttion," 2 nd the Deprtment of Veterinry Microbiology nd Prsitology,3 School of Veterinry Medicine, Louisin Stte University, Bton Rouge, Louisin Received 3 September 1986/ccepted 16 December 1986 Comprisons of peptide nd oligonucleotide mps of glycoproteins nd RN from nine isoltes of equine infectious nemi virus (EIV) tht were generted during prllel infections of two Shetlnd ponies reveled tht ech isolte ws structurlly unique. Ech EIV isolte contined unique subset of vrint peptides, oligonucleotides, or both, indicting tht structurl vrition in EIV is rndom nd noncumultive process nd tht lrge spectrum of possible EIV vrints cn be generted in infected nimls. The members of the lentivirus subfmily of retroviruses, including equine infectious nemi virus (EIV), visn virus, nd humn immunodeficiency virus (HIV), cuse chronic diseses in vriety of nimls nd humns (2, 5, 6, 8, 16). The persistent infections cused by these viruses in some instnces cn be trced to their bility to circumvent or overcome the immune responses of the host (4, 6, 9, 18). In the cse of EIV, which cuses unique episodic disese in horses, it hs been demonstrted tht isoltes recovered from n infected niml during different febrile episodes cn be distinguished ntigeniclly nd structurlly by vriety of biochemicl nd immunologicl ssys (11, 15, 18). Results of these studies hve indicted tht structurl nd ntigenic vrition is loclized to the envelope glycoproteins gp9o nd gp45 (11, 18). The occurrence of envelope glycoprotein vrition hs lso been demonstrted for visn virus nd HIV (1, 4, 7, 19). criticl question regrding lentivirus vrition is the number of vrints tht cn occur in nture, mjor fctor in ssessing potentil vccines. To nlyze the spectrum of structurl vrition mong isoltes of EIV, we used peptide, glycopeptide, nd oligonucleotide mpping procedures to compre nine EIV isoltes recovered during distinct clinicl episodes in two ponies infected with the sme virus. To conduct these experiments, identicl virulent virus inocul were used in prllel infections of two Shetlnd ponies s described previously (11, 13, 18). The clinicl histories of these nimls re summrized in Fig. 1. Plsm smples tken during clinicl episodes were subjected to endpoint dilution in fetl equine kidney cells to recover the predominnt virus popultion (13). The nine EIV isoltes recovered were propgted in fetl equine kidney cells nd purified by glycerol grdient centrifugtion (10, 11, 14). Ech virus isolte ws determined to be ntigeniclly distinct by using vriety of immunossys employing serum from infected ponies, pnels of monoclonl ntibodies, or both * Corresponding uthor. t Louisin griculturl Experiment Sttion pper no t Present ddress: Lovelce Inhltion Toxicology Reserch Institute, lbuquerque, NM (18;. Orrego, Ph.D. thesis, Louisin Stte University, Bton Rouge, 1983). The extent of structurl vrition in the virl glycoproteins gp9o nd gp45 for ech of the nine EIV isoltes ws ssyed by two-dimensionl 125I-lbeled tryptic peptide (11, ) 0. H- cl - 0) -_.0.. Doys Post Inoculotion r(i re5.. FIG. 1. Clinicl histories of experimentlly infected ponies showing clinicl episodes from which virus isoltes were recovered. Both nimls received identicl.intrvenous virus inocul contining 4.8 loglo 50% tissue culture infective doses of host-dpted EIV (13). ll sustined rectl temperture recordings bove 39 C (dshed line) were considered bnorml nd were designted febrile episodes. () Pony 91 exhibited clssicl chronic equine infectious nemi tht ws chrcterized by recurring febrile episodes. Pony 91 died fter four febrile episodes, on dy 180. (B) Pony 127 lso experienced chronic equine infectious nemi. The 5 months between the third nd fourth febrile episodes represent n inpprent stge of the disese in which the symptomtic niml remins virus cmer. 1266

2 VOL. 61, 1987 NOTES B *0* b4 0 lm I0 eo P O02 Cj U ftm 23 U onz sbt b 60 lo*' 'ic2 310@ @ O ! ,, U~~~~~~~'O FIG. 2. Composite two-dimensionl tryptic peptide ptterns generted from mps of 251I-lbeled glycoproteins gp9o nd gp45 from ech EIV isolte. The peptides generted for ech virus isolte were mpped in duplicte. The composite mps were generted by compring ech virus isolte with ll other isoltes by directly overlying utordiogrphs. rrows indicte the directions of electrophoresis () nd chromtogrphy (b). rbitrry numbers were ssigned to ech peptide for identifiction. Closed circles indicte peptides tht were common to ll virus isoltes. Open circles indicte vrint peptides. The htched re represents rdioiodinted tryptic glycopeptides tht were not resolved by stndrd peptide mpping techniques (11). () gp9o composite peptide mp. (B) gp45 composite peptide mp. Q- O" OU 0 lso o3 18) nd glycopeptide (17) mpping procedures. ll peptide nd glycopeptide ptterns were reproducible on repeted mppings, nd virus isoltes remined stble during continued pssge in tissue culture. Figure 2 shows the composite mps developed to disply conserved nd vrint peptides for gp9o nd gp45. totl of 54 peptides were identified on comprison of the gp9o component of the nine vrints nd the prototype strin of EIV. Twenty-seven (50%) of the gp9o peptides were found to be common to ll isoltes. The pttern of vrition of the remining peptides ws used to uniquely identify eight of nine vrint strins, s well s the prototype strin of the virus (Tble 1, gp9o peptides). Only isoltes P3.2-2 nd P3.2-3 could not be distinguished by gp9o peptide mpping nlysis. For gp45 only 11 of 45 peptides (24%) were common to ll isoltes. In this cse the pttern of vrint peptides could be used to distinguish ll isoltes (Tble 1, gp45 peptides); P3.2-2 nd P3.2-3 could be distinguished. Tble 1 lists those peptides not common to ll virus isoltes. Some of these vrint peptides were present in isoltes from only one pony. These included gp9o peptides 12, 18, 20, nd 25 nd gp45 peptides 6, 14, 15, 25, 27, 29, nd 30. gp9o peptides 18 nd 25 were unique to single virus isoltes. Most other peptides were present in nd vried mong isoltes from both nimls, however. For exmple, gp9o peptide 49 ws present in P3.1-2, P3.2-1, nd P3.2-4; gp9o peptide 52 ws present in P3.1-1, P3.1-3, P3.2-2, P3.2-3, nd P The dt suggest tht structurl vritions mong virus isoltes do not ccumulte with time, s peptides pper in one isolte, only to be lost in subsequent isoltes. Insted, it ppers tht limited set of peptides vry independently, leding to the presence of different subset of peptides for ech isolte. In contrst to the envelope glycoproteins, the peptide mps for the virl core proteins p9, p15, nd p26 were identicl for ll virus isoltes (dt not shqwn), s reported previously (11, 17). gp9o nd gp45 from ech virus isolte were lso nlyzed by glycopeptide mpping to compre the glycosylted tryptic peptides not resolved by stndrd peptide mpping procedures (11, 17, 18). Four clsses of gp9o nd two clsses of gp45 glycopeptide ptterns were observed. Representtive glycopeptide mps re shown in Fig. 3, nd in Tble 2 the glycopeptide ptterns obtined for ech virus isolte re summrized. These dt indicte tht chnges in the pttern of glycosyltion of gp9o nd gp45 re independent of one nother. For exmple, ll virus isoltes from pony 91 (P3.2-1 through P3.2-4) shre common gp45 glycosyltion pttern, while they exhibit four different gp9o glycosyltion ptterns. It is interesting tht virus isoltes P3.2-2 nd P3.2-3, which produce pprently identicl gp9o peptide ptterns, exhibit different glycopeptide mps. To nlyze the genomic vrition mong EIV isoltes, RN ws purified from ech virus nd nlyzed by oligonucleotide mpping by previously described techniques (15, 18). composite mp showing conserved nd vrint oligonucleotides ws then developed (Fig. 4). totl of 51 oligonucleotides re represented in Fig. 4, nd 33 (65%) of these were common to ll isoltes. Eight of the nine virus strins nd the prototype virus could be distinguished by nlysis of the distribution of vrint oligonucleotides. Only isoltes P3.2-1 nd P3.2-4 could not be distinguished esily. (Tble 3). s observed with the peptide mps described bove, there ppered to be no ccumultion of vrint oligonucleotides mong lte virus isoltes. Results of the experiments presented here provide

3 1268 NOTES detiled comprison of nine virus isoltes recovered from two ponies infected with identicl inocul of EIV. Severl importnt properties of EIV vrition cn be concluded from these observtions. distinct virus popultion predomintes during ech febrile episode in persistently infected pony. The vrint virus strins exmined in this study were recovered from endpoint dilutions of plsm nd, thus, re ssumed to represent the predominnt virus popultions in the infected niml t the time the plsm smples were obtined. There ppers to be reltively lrge number of structurl vritions possible in EIV. In ddition to the nine virus isoltes described here, we recovered five more EIV isoltes from third pony tht received the sme initil virus inoculum (unpublished dt). These virus isoltes could lso be distinguished structurlly, bringing the totl number of unique vrints generted from this virus inoculum to 14. These 14 isoltes were distinct from isoltes recovered from L. 1I [if IV 1I B J. VIROL. TBLE 1. Distribution mong EIV strins of vrint peptides from the envelope glycoproteins gp9o nd gp45 Distribution of: Virus Peptide gp9o peptides gp45 peptidesb Prototype 7, 20, 28, 36, 44, 45, 46, 47, 49, 50, 51, 52, 53 P , 15, 28, 29, 30, 33, 35, 36, 38, 39, 40, 44, 45, 46, 47, 51, 52, 53 P , 12, 15, 21, 29, 35, 36, 38, 39, 43, 46, 47, 48, 49, 50, 51 P , 15, 25, 28, 29, 30, 33, 35, 39, 44, 45, 46, 47, 52, 53 7, 12, 15, 29, 30, 35, 38, 39, 43, 46, 47 P , 28, 36, 38, 39, 40, 44, 45, 46, 49, 51, 53 P , 15, 20, 21, 28, 29, 30, 36, 38, 39, 40, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53 P , 15, 20, 21, 28, 29, 30, 36, 38, 39, 40, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53 P , 15, 18, 20, 21, 28, 29, 30, 36, 38, 39, 40, 43, 44, 45, 46, 47, 49, 50, 51, 53 P , 20, 21, 28, 29, 30, 33, 35, 40, 43, 44, 45, 47, 48, 50, 51, 52, 53 Prototype P3.1-1 P3.1-2 P3.1-3 P3.2-1 P3.2-2 P3.2-3 P3.2-4 P3.2-5 I Compre with Fig. 2. b Compre with Fig. 2B. 3, 6, 12, 17, 21, 24, 26, 35, 36, 39, 41 2, 3, 21, 22, 23, 30, 31, 33, 35, 41, 42, 44, 45 2, 3, 10, 18, 20, 22, 23, 29, 33, 38, 41, 42, 44, 45 2, 10, 11, 12, 17, 18, 20, 21, 22, 23, 24, 26, 33, 35, 36, 38, 39, 41, 42, 43, 44, 45, 46 2, 3, 10, 17, 18, 20, 22, 23, 29, 33, 36, 39, 41, 42, 44, 45 3, 6, 11, 12, 18, 19, 21, 23, 24, 26, 35, 36, 39, 41, 42, 43 2, 10, 11, 12, 15, 18, 19, 21, 24, 25, 27, 30, 35, 39, 42, 45 2, 6, 10, 11, 12, 14, 15, 17, 18, 21, 22, 23, 24, 27, 30, 36, 39, 41, 43, 44, 45, 46 6, 10, 11, 12, 18, 21, 23, 24, 26, 30, 35, 36, 38, 39, 42, 44, 45, 46 2, 6, 10, 11, 12, 14, 18, 20, 21, 22, 24, 25, 26, 27, 30, 33, 35, 36, 38, 39, 42, 44, 45, 46 FIG. 3. Exmples of glycopeptide ptterns obtined for lectinpurified 'l25-lbeled gp9o nd gp45 glycopeptides. For nlysis of these ptterns, the presence or bsence of verticl groups of glycopeptides, s opposed to individul glycopeptides were compred, such tht possible microheterogeneity of the crbohydrte moiety did not interfere with the comprisons (17). () gp9o glycopeptide mps. Clss I, P3.2-1; clss II, P3.2-2; clss III, P3.1-3; clss IV, P3.2-5 (B) gp45 glycopeptide mps. Clss I, P3.1-1; clss II, P For ll mps the direction of electrophoresis ws from left to right; the direction of chromtogrphy ws from bottom to top. independent seril trnsmissions of EIV between ponies (11, 15). Thus, totl of t lest 17 distinct structurl vrints were ctloged in our lbortory. This lrge rnge of EIV vrition is similr to tht observed for HIV, in which no two virus isoltes exmined hve been found to be identicl in restriction enzyme mpping, DN sequencing studies, or both (1). The evolution of EIV vrints during persistent infection is evidently rndom; i.e., no predictble sequence of virus vrition ws observed in the two experimentlly infected nimls, lthough ech niml received identicl virus inocul. This result differs from the dt reported for prllel persistent infections with visn virus, in which similr ptterns of vrint evolution were observed (3). This difference my reflect tht lrger spectrum of vrition is possible in EIV compred with visn virus. TBLE 2. Clssifiction of EIV isoltes by glycopeptide ptterns of the envelope glycoproteins gp90 nd gp45 Glycoprotein Viruses in: Clss I Clss II Clss III Clss IV gp9o Prototype P3.1-1 P3.1-3 P3.2-3 P3.2-1 P3.1-2 P3.2-5 P3.2-4 gp45 Prototype P3.1-2 P3.1-1 P3.1-3 P3.2-1 to P3.2-5

4 VOL. 61, 1987 NOTES 1269 b D27 _ Q4S 25 * Q C 40 1iO Q0(n @ * , o4o FIG. 4. Composite oligonucleotide fingerprint generted by comprisons of oligonucleotide mps from EIV isoltes. For purposes of comprison, virl RN from ech virus isolte ws purified nd subjected to mpping procedures on t lest two seprte occssions, nd typiclly three to four mps were used for mking comprisons between ny two isoltes. Comprisons between ll isoltes were mde by directly overlying utordiogrphs. The composite reflects only the high-moleculr-weight (lower) region of the originl mps. rbitrry numbers were ssigned to ech oligonucleotide for identifiction. Closed circles indicte oligonucleotides tht were common to ll virus isoltes. Open circles indicte vrint oligonucleotides. The directions of first-dimension () (8% polycrylmide; ph 3.3) nd second-dimension (b) (22% polycrylmide; ph 8.2) electrophoresis re indicted by rrows. The vritions in EIV genomic RN nd glycoproteins re not necessrily cumultive. Subsequent virus strins my not rise from the virus strin which predomintes in the preceding febrile episode. Rther, it ppers tht rndom muttions generted during virus repliction result in vriety of integrted proviruses, ny one of which my led to the genertion of future predominnt virus strins. In contrst to EIV, sequentil isoltes of visn disply cumultive chnges (3). Sequentil HIV isoltes like EIV, however, fil to disply cumultive chnges. Noncumultive vrition of HIV hs been interpreted to indicte prllel evolution of vrints in the infected individul (7). n lterntive explntion for the results obtined for both EIV nd HIV would be high muttion rte which msks ny direct linege between sequentil isoltes. The time required for the evolution of vrint popultions of EIV is vrible but cn be remrkbly rpid. The shortest time observed between clinicl episodes nd distinct EIV isoltes ws pproximtely 15 dys (P3.2-1 to P3.2-2). In contrst, the genertion of vrints of visn virus Virus TBLE 3. Distribution of vrint oligonucleotides Oligonucleotide Prototype 1, 4, 6, 13, 15, 16, 19, 32, 41, 47 P , 4, 6, 13, 16, 19, 32, 36, 40, 47 P , 4, 6, 16, 19, 32, 36 P , 4, 6, 13, 16, 19, 32, 36, 46, 47 1, 4, 6, 13, 16, 19, 32, 33, 36 P3.2-1 (1), 3, (4), 6, 13, 16, 19, 32, 36, 47 P , 6, (11), 18, 19, 32, (36) P , 18, 37, 47 P , 3, 4, 6, 13, 16, 19, 32, 36, 47 P , 6, 11, 13, 18, (19), 22, 32, (36), 48 Prentheses indicte the presence of wek signl in the position of the oligonucleotide for tht virus isolte. usully requires t lest yer (12), nd the genertion of HIV vrints is believed to follow similr time course (7). In this regrd, EIV provides unique model to study rpidly chnging virus structure, s well s the dynmic interction between host immune responses nd evolving lentivirus ntigens. This work ws supported by the Louisin griculturl Experiment Sttion, the Louisin Stte University School of Veterinry Medicine, nd Public Helth Service grnt C from the Ntionl Cncer Institute. LITERTURE CITED 1. lizon, M., S. Win-Hobson, L. Montgnier, nd P. Sonigo Genetic vribility of the IDS virus: nucleotide sequence nlysis of two isoltes from fricn ptients. Cell 46: Brre-Sinoussi, F., J. C. Chermnn, F. Rey, M. T. Nugeyre, S. Chmret, J. Gruest, C. Duguet, C. xler-blin, F. Brun- Vezinet, C. Rouzioux, W. Rozenbum, nd L. Montgnier Isoltion of T-lymphotropic retrovirus from ptient t risk for cquired immune deficiency syndrome (IDS). Science 220: Clements, J. E., N. D'ntonio, nd 0. Nryn Genomic chnges ssocited with ntigenic vrition of visn virus. II. Common nucleotide sequence chnges detected in vrints from independent isoltions. J. Mol. Biol. 158: Clements, J. E., F. S. Pedersen, 0. Nryn, nd W.. Hseltine Genomic chnges ssocited with ntigenic vrition of visn virus during persistent infection. Proc. Ntl. cd. Sci. US 77: Gllo, R. C., P. S. Srin, E. P. Gelmnn, M. Robert-Guroff, E. Richrdson, V. S. Klynrmn, D. Mnn, G. D. Sidhu, R. E. Sthl, S. Zoll-Pzner, J. Leibowitch, nd M. Popovic Isoltion of humn T-cell leukemi virus in cquired immune deficiency syndrome (IDS). Science 220: Hse,. T Pthogenesis of lentivirus infections. Nture (London) 322: Hhn, B., G. Shw, M. Tylor, R. Redfield, P. Mrkhm, S. Slhuddin, F. Wong-Stl, R. Gllo, E. Prks, nd W. Prks Genetic vrition in HTLV-III/LV over time in ptients with IDS or risk for IDS. Science 232: Issel, C. J., nd L. Coggins Equine infectious nemi: current knowledge. J. m. Vet. Med. ssoc. 174: Kono, Y., K. Kobyshi, nd Y. Fukung ntigenic drift of equine infectious nemi virus in chroniclly infected horses. rch. Gesmte Virusforsch. 41: Montelro, R. C., N. Lohrey, B. Prekh, E. W. Blkeney, nd C. J. Issel Isoltion nd comprtive biochemicl properties of the mjor internl polypeptides of equine infectious nemi virus. J. Virol. 42: Montelro, R. C., B. Prekh,. Orrego, nd C. J. Issel ntigenic vrition during persistent infection by equine infectious nemi virus, retrovirus. J. Biol. Chem. 259: Nryn, O., J. E. Clements, S. Kennedy-Stoskopf, nd W. Royl, III ntigenic vrition in the lentiviruses tht cuse visn-medi in sheep nd rthritis-encephlitis in gots, p In T. H. Birkbeck nd C. W. Penn (ed.), ntigenic vrition in infectious diseses. IRL Press, Wshington, D.C. 13. Orrego,., C. J. Issel, R. C. Montelro, nd W. V. dms, Jr Virulence nd in vitro growth of cell-dpted strin of equine infectious nemi virus fter seril pssge in ponies. m. J. Vet. Res. 43: Prekh, B., C. J. Issel, nd R. C. Montelro Equine infectious nemi virus, puttive lentivirus, contins polypeptides nlogous to prototype-c oncornviruses. Virology 107: Pyne, S., B. Prekh, R. C. Montelro, nd C. J. Issel Genomic ltertions ssocited with persistent infections by equine infectious nemi virus, retrovirus J. Gen. Virol. 65: Popovic, M., M. G. Srngdhrn, E. Red, nd R. C. Gllo.

5 1270 NOTES Detection, isoltion, nd continuous production of cytopthic retroviruses (HTLV-IIL) from ptients with IDS nd pre-ids. Science 224: Slinovich, O., nd R. C. Montelro Comprison of glycoproteins by two-dimensionl mpping of glycosylted peptides. nl. Biochem. 157: Slinovich, O., S. L. Pyne, R. C. Montelro, K.. Hussin, J. VIROL. C. J. Issel, nd K. L. Schnorr Rpid emergence of novel ntigenic nd genetic vrints of equine infectious nemi virus during persistent infection. J. Virol. 57: Wong-Stl, F., G. M. Shw, B. H. Hhn, S. Z. Slhuddin, M. Popovic, P. Mrkhm, R. Redfield, nd R. C. Gllo Genomic diversity of humn T-lymphotropic virus type III (HTLV-III). Science 229: