Characterization of variable regions in the envelope and S3 open reading frame of equine infectious anemia virus.

0022-538X/91/084255-08$02.00/0

CopyrightC 1991,American Society for Microbiology

Characterization of Variable Regions in

the Envelope and S3 Open

Reading

Frame

of Equine

Infectious

Anemia Virus

SOREN ALEXANDERSEN*ANDSUSAN CARPENTER Department of Veterinary Microbiology andPreventiveMedicine,

IowaState University, Ames, Iowa50011

Received 11 February 1991/Accepted 30April 1991

Thepolymerase chain reactionwasusedtoamplifyand clonepartsofthe envelopegeneandoverlappingS3 open reading frame, thought to encode rev, of the virulent in vivo-derived Th-1 isolateof equine infectious

anemia virus(EIAV).The results indicated that EIAV consists ofaheterogeneousmixture ofgenotypespresent

atthe firstfebrile cycle after initial infection. We showedthat the Th-lisolateapparentlycontains nondefective

genotypes aswellastypes which have transmembrane protein truncationsor are revdeficient. Furthermore,

wecouldconfirmthepresenceofahypervariable region in the gp9Oenvelopeglycoprotein. Taken togetherwith

earlier data on the heterogeneity ofthe regulatory motifs present in the long terminal repeat sequences of

viruses from the same in vivo isolate (S. Carpenter, S. Alexandersen, M. J. Long, S. Perryman, and B.

Chesebro,J. Virol. 65:1605-1610, 1991),ourfindings indicate that EIAVusesacomplexsystemof diversity

inbiological phenotypes togetherwithvariationin regulatory andantigenicmakeuptoevade hostresponseand

tocausepersistent infection and recurrentchronic disease.

Equineinfectious anemia virus (EIAV) causes a persistent

infection in horses. The infection is characterized by a

clinically variable disease course which can be acute,

chronic,orasymptomatic (27). EIAV belongs to the

subfam-ilyLentivirinae of the family Retroviridae and is genetically

and antigenically related to other lentiviruses, including human immunodeficiency virus (HIV) (11, 24, 28, 41, 43, 53, 62).

Lentiviruses vary considerably, and infected individuals

or animals can harbor a genetically complex mixture of

genotypes presentatthe sametimeor overperiods of time

(4, 7, 9, 10, 14, 20, 21, 25, 28, 36, 40, 44, 48, 55, 56, 62, 64). One factor important in lentivirus variant selection is the

specificity ofthe host immune response (13, 32, 38, 42, 44).

Inaddition, recent findings suggest that variation in

biolog-ical phenotype is an important mechanism of lentivirus

pathogenesis. Progression todisease in individuals infected with HIV could be correlated with recovery of virus isolates which were more cytopathic and exhibited a broader in vitro

host range (9, 64). Similarly, HIV isolates recovered from

asymptomatic individuals replicate slowly and to low titers in vitro, whereas isolates recovered from individuals with

more severe clinical signs ofinfection arecharacterized by

rapid replication and production of high levels of reverse

transcriptase activity (4, 20).

Progressin defining moleculardeterminants important in

viral virulence and host response is beginning to emerge.

Recentstudiessuggestthat severalviral genes,aswellasthe

long terminal repeat (LTR), contribute to the biological

diversity of HIV type 1 (23, 65). Also, the presence of

specific enhancer elements in other retrovirus LTRs has

been shown toinfluence cell tropism, virulence,and

leuke-mogenicity(33, 58). We havepreviouslyshown that theLTR

sequences of an avirulent cell culture-adapted isolate of

EIAVdiffered from the invivo parental isolate (6). Thetwo

isolates differed in the target cell tropism, i.e.,the parental

isolate Th-1 grew well inmacrophagesandpoorly inequine

*

Corresponding

author.

dermalcells, whereas theoppositewastruefor the

culture-adaptedisolate MA-1 (7). Moreover, thespecific regulatory

sequencemotifs present in the LTRs of thetwoisolateswere

significantly different (6). The retrovirus LTR contains the

sole promoterfor viraltranscriptionandisregulatedbothby

viral trans-acting proteins such as tat (3, 60, 61) and by a

multitude ofcellularproteins involved in DNA binding and

transcription complex assembly, initiation, and elongation

(16, 46). Theseresultssuggested that variation in regulatory

sequences in the LTRmay be important in EIAV host cell

tropismand pathogenesis in vivo.

Instudies of EIAV and visnavirus, genetic mutations in

the viral env gene were associated with the occurrence of

antigenicvariant viruseswhichwerepostulatedtopersist by evasion ofthe host immune response (12, 13, 42, 48). For

HIV, the different genotypes have been shown to vary in

biological phenotype, and this variation, in part, is

deter-minedbysequences in theviral envelope(21,65). Also,the

difference in cell tropism ofHIV has been mapped to the

envelope, particularly sequences near the hypervariable

loop V3(21, 35, 45).

In the presentstudywecharacterizedtheenvelope

region

andoverlapping S3 open reading frame, thoughttoencode

rev, of the Th-1 isolate of EIAV derived directly from

infected horse macrophages after asinglepassage in vitro.

The results indicate thatEIAV,asdescribed aboveforHIV,

consists ofa heterogeneous mixture ofgenotypes

already

present at the first febrile cycle after initial infection. We

could show that the isolateapparently contains nondefective

genotypes as well as types which have transmembrane

protein truncations or are rev deficient. Furthermore, we

were able to confirmthepresence ofa hypervariable region

in the gp9O envelopeglycoprotein previously

suggested by

Payne etal. (48). Togetherwith ourearlier dataon

hetero-geneityin the LTRsequencesfrom the samein vivo isolate

(6),thesefindingsindicate thatEIAVuses a

complex

system

ofdiversityinbiological phenotypes

together

withvariation

in regulatory andantigenic makeupto evade host response

and to cause

persistent

infection and recurrent chronic disease.

4255

on November 10, 2019 by guest

http://jvi.asm.org/

TABLE 1. Nucleotide positions and primersequencesfor variousregions ofthe EIAVgenomea Sequence

Region

5'Primer 3' Primer

5310 5903

S2/gp9O 5'-ACATGGTCAGCATCGCATTC-3' 3'-ATTGAGACATGCACAACTCC-5'

5730 6413

gP9O 5'-GAGACGTGGGCAAGAGTGAA-3' 3'-ATACTTTGTTTGGTCCATGT-5'

7264 7673

gp45/S3 5'-TGGTGCAGGGTCCTCCGGCA-3' 3'-TCGGGTTCTTCCTTGAGAGC-5'

a Thenucleotide positionof the5'endisgivenabove each sequence.

MATERIALSANDMETHODS

Virus and cells. Th-1 is a field-derived isolate of EIAV

recovered from a horse experimentally inoculated with

whole blood from anEIAV-seropositive, naturally infected

horse (7, 8). The experimentally infected horse was

moni-tored daily after infection, and primary horse macrophage

cultures (HMC) were established from peripheral blood

mononuclearcells aspreviously described (7, 17). The Th-1

virus stock is the cell-free supernatant fluid from these

primary HMCestablished duringthefirstfebrile episode of

equine infectious anemia and grown for 8 to 10 days in

culture (6, 7, 17). Fresh HMC were established from an

EIAV-negative horse, and this culture was inoculatedwith

the Th-1 virus stock. This culture was grown for 6 days,

tested, and foundpositivefor reverse transcriptaseactivity,

and total DNA wasextracted andanalyzedbySouthernblot

hybridization as previously described (6). EIAV sequences

in this DNA preparation represent infectious EIAVafter a

single passage in HMC and, thus, should accurately typify

EIAVgenotypes present in vivo.

Molecular cloning. Amplification and cloning of LTR

se-quences from the DNA preparation ofTh-1-infected HMC

have previously been briefly described (6). In the present study we amplified and cloned segments of the viral

enve-lopeand overlapping S2 and S3 open reading frames using

the same Th-1 DNApreparation and similar techniques. To

clone Th-1 sequences, specific segments were amplified by

thepolymerase chainreaction (PCR) using 20-mer

oligonu-cleotide primers complementaryto sequences atthe 5' and

3' borders of the segment to be amplified. Primers were

synthesizedwith an AppliedBiosystems 380B DNA

synthe-sizer, and the sequence was based on the published

se-quenceof the MA-1 isolate of EIAVderived by cell culture

passage of the Th-1 stock (6). All numbering of nucleotides

in the present report is based on the available GenBank

sequenceof EIAV Wyoming (14, 28), except that a G is put

inafternucleotide 5348 to make the env open reading frame

continuous, aspreviouslydescribed (53). This numbering is in accordance with our previously published sequence of the MA-1isolateof EIAV (6). The nucleotide positions, 3' and 5' ends, and sequences of the primers used for the different areasof the EIAV genome are shown in Table 1.

PCRs were performed in a volume of 100

,u1

containing 200

ng of each oligonucleotide primer, 50 mM

KCl,

10 mM

Tris-HCl (pH 8.4 at 20°C), 1.5 mM

MgCl2,

20

p.g

ofgelatin, 0.2 mM each dNTP, 5 U of Taq DNA polymerase (Perkin

Elmer Cetus, Norwalk, Conn.), and 30 ng of DNA to be

amplified. The reactions were amplified for 25 cycles at an

annealingtemperature of 47°C for 2 min, a synthesis temper-atureof72°C for1.5min, and a heatdenaturation

tempera-tureof94°C for 1min

by using

anautomatedthermal

cycler

(JSP

EL-Service

APS,

Glostrup, Denmark). Amplified

DNA

was partially purified

by

phenol-chloroform extraction and

ethanol

precipitation.

Formostof the

clonings,

XbaIlinkers

wereadded and the DNAwasligatedtopGEM3Z

(Promega

Biotec,

Madison, Wis.) and used to transform competent

Escherichia coli JM109 as described previously

(2, 6).

For

cloning

ofthe

S2/gp9O

segment (clone series 5a), the DNA was cut with BamHI

(cuts

at nucleotide 5338) and with

HindIII (cuts at nucleotide 5780) and cloned into

pUC19

(GIBCO BRL,

Gaithersburg, Md.). Colonieswere screened

for the presence of an EIAV insert by hybridization to

EIAV-specific probes

radiolabeled as described

previously

(6).

The segment of gp9O amplified with the primers from

nucleotide 5730 to6413 hasaninternal XbaI site at

nucleo-tide6161 andwas thus clonedastwofragments

(designated

clone series 7a and

7b).

Fiveto sevenindependent

positive

colonies from each cloned segment, a total of 26

clones,

were picked, purified by replating, and sequenced by the

dideoxynucleotide chain termination method(57) with

dou-ble-stranded DNA templatesas described previously (2,

5,

6). Each setofclones, representinga specific segment, was

cloned from a single PCR experiment. Cloningofthe

gp9O

segment from another PCR experiment showed the same

distribution of sequence differences (datanot

shown).

Sequences were analyzed by using the Beckman

Micro-genieand the IBI Pustell Sequence Analysis Software

pro-grams. Genetic comparisons were based on sequences of

EIAV MA-1 (6) and EIAVWyoming available in GenBank

orinprevious publications (14, 28, 48, 53, 62).

Nucleotide sequence accession numbers. GenBank

acces-sion numbers for thesequences reported hereareM62655to

M62680.

RESULTS AND DISCUSSION

Wepreviously identified regions in the EIAVenvregion in

which the MA-1 isolatediffered from the Wyoming isolate of

EIAV(6). In the present studyweused PCRtoamplify and

clone theseregionsfrom the Th-1isolate,theparental stock

ofMA-1 whichbiologically differs in cell tropism and

viru-lence(7).Th-1 sequenceswereamplified and cloned

directly

from HMCestablished fromaninfected horse during the first

febrilecycle after infection. Areas of the env gene,including

major parts of the surface gp9O and transmembrane

gp45

glycoproteins previously described to have a considerable

degree of variability,werecloned andanalyzed (6, 48). One

region of gp9O analyzed overlaps with the S2 openreading

framehavinganas-yet-unknownfunction. The region of the

gp45 analyzed overlaps with the S3 open reading frame

thoughtto encoderev(15, 51,63).

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.67.569.80.188.2]

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 '0 0 10 0 t0 0 10 0

T C') C') It It 0

0 0 0 0 0 0

0 0 0 0 0 0

'0 0 0 '0 0

10 10 tN OD

l1111111l 1111ll1l 11l 11l

[image:3.612.60.552.59.334.2]

U

LTR

FIG. 1. Schematic representation of theEIAVprovirus. All numbering of nucleotides inthe present report is based onthe available

GenBanksequenceof EIAV Wyoming (14, 28),exceptthataG isputin after nucleotide5348tofixtheenvopenreading frame(53). The total proviralgenome, including bothLTRs,is thus8,229nucleotides. The locations of thetwocomplete LTRs (nucleotides 1to321and7909to

8229) and theopenreading framesgag(nucleotides387to1922),pol (nucleotides1682to5119),env(nucleotides 5303to7888),S1 (nucleotides 5123to5272), S2 (nucleotides 5283to5480), and S3 (nucleotides7234to7638)areindicated. S3 isinreadingframe1;pol, S1, andenvare

inreading frame 2; andgagand S2arein reading frame3. Theputative cleavage siteatnucleotide6643in theenvgeneis indicatedasavertical arrow.Regionsof Th-1 whichwecloned andsequencedareindicatedbythickhorizontal bars. Sequencedifferencesamongthe MA-1(6)and Th-1 clones are shown as thin vertical lines underneath the thick horizontal bars. Fordetails on sequence differences see Fig. 2. The hypervariable region, described previously byPayneetal.(48),spanning nucleotides6176to6343is shownas ahatchedhorizontal bar. The

twostopsignalsin theS3openreadingframe of Th-1 clones 8a51 and8all3areshownashatched circles,and thestopsignalingp45of Th-1 clone8a81isshownas afilled circle.

Nucleotide sequencecomparison betweenthe Th-1 and the

MA-1isolates of EIAV.PCR-generatedclones from the Th-1 isolate of EIAV were cloned and nucleotide sequenced.

Regionsweanalyzedandnumbers of individual cloneswere

as follows: one region in the S2 open reading frame/gp9O

overlap (seven independentclonesdesignatedseries5a),two

regionsinthegp9O area(seven cloneseachofseries 7a and

b), and one region in the gp45/S3 area (five independent

clonesdesignatedseries8a). Oligonucleotide-primer binding sites and regions near the primers which are difficult to

sequence in both orientations were excluded from further

analysis. Thegenomic organization ofEIAV and the

posi-tions of the sequenced segments of the Th-1 isolate are

shown in Fig. 1.

Wecomparedthe nucleotidesequencesof the Th-1 clones

with that of the cellculture-adapted MA-1 isolate of EIAV derived from the Th-1 stock(6) and found severalchanges

(Fig.1 and2).Of the1,029nucleotidessequencedinthegp9O

areaof theenvgene, 16 nucleotides(1.6%)differed between

the Th-l-related clones (Th-1 and MA-1). In the gp45/S3

region,370 nucleotidesweresequencedand 13 bases(3.5%)

differed. AscanbeseenfromFig. 1and2,thechangeswere

notevenlydistributed butweremainlyfound intworegions

of thegp9Oand intwoorthree regionsof thegp45/S3 open

reading frame. No changes were found in the S2 open

readingframe. Of the sevenclones analyzedin the

amino-terminal partofgp9O (series 5a),five cloneswereidenticalto each otherand also identicaltoMA-1. Theothertwoclones in this region had a single nucleotide change each, a

vari-abilitybetweenanypairof the clones between 0 and 2 of444

(Oto0.5%)nucleotides. In the centralregionofgp9O (series

7a) four clones were identical to each other and toMA-1. Theother three clones had 1or2nucleotidechanges each,a

variability ofanypair of clonesbetween 0 and 4 of 373 (0to

1.1%) nucleotides. In the region close to the

carboxy-terminal endofgp9O (series 7b), onlythree ofsevenclones

wereidentical and these three clones hadasinglenucleotide

change comparedwithMA-1 (Fig. 2). Thehighest degreeof

variabilityofanypairof clones in thisregion (series 7b)was

5changesof 212nucleotidesanalyzed,whichisavariability

of 2.4%. Inthegp45/S3 region analyzed (series 8a), onlytwo of the five clones were identical. However, these clones

werehighly differentfrom MA-1 (10 changesof 360 [2.8%]

nucleotidesanalyzed)andwerealsohighlydifferentfrom the other Th-1clones(8to10 nucleotidedifferences).The other three clones in thisregionweremoresimilartoMA-1and to

each other, i.e., 1 to 5 nucleotide differences between any

pair (0.3 to1.4%variability).

Analysisofpotential coding changes. Analysisof thecoding

potential of the Th-1 clones revealed several interesting

features. First,the existence andpositionofahypervariable

region in thegp9O region ofenv, suggested by Payne et al.

LTR

on November 10, 2019 by guest

http://jvi.asm.org/

gp9O region of envelope

Clone

NA-1 5a3 5.8 5.9 5.10 5a11 5.12 5a13

Nucleotide

5743 G (Met) A(lie)

5762 G (Ata) A(Thr*)

MA-1 7a41 7a51 7a71 7.81 7a91 7al16 7al18

Nucteotide

5838 5895 5966

6062

6150

A(Gin)

G (Arg)

T (Phe)

A(Lys) T(lie)

T (Leu)

T(Leu)

C(Leu) G(Gtu)

A (Asn*)

MA-1 7b41 7b51 7b71 7b81 7b91 7b116 7b1i1

Nucleotide

[image:4.612.150.484.81.450.2]

6373 G (Met) A (lie)

FIG. 2. Characterization of nucleotide and amino acid differences between MA-1 and Th-1 clones. The differencesinnucleotideand amino acid sequences between the MA-1 isolate of EIAV (6) and the sequenced Th-1 clones are indicated. For details on the sequence and numbering of EIAV MA-1, see reference 6. The regions of the Th-1 isolate analyzed are as follows: gp9O/S2 region, clone series 5a, nucleotides 5337to5780; gp9Oregion, clone series 7a, nucleotides 5789to6161, and clone series 7b, nucleotides 6162to6373;gp45/S3 region, cloneseries8a, nucleotides 7284to7643. Nucleotide changesoccurringinthepotentialcysteine-cysteine loop regionfrom nucleotide 6176

to6343suggestedtobehypervariableby Payneetal.(48)areboxed,asarethecoding changes givingprematurestopcodons.*,amino acids potentially involved inglycosylation. Two changed N-linkedglycosylation sitesareunderlined.

(48), could be confirmed. Second, the data indicated the

presence of another variable region in thegp9O protein and

also provided evidence, as previously suggested byus (6),

that the amino-terminal portion of gp9O and the S2 open

reading frame are highly conserved. Third, clones having

premature stop codons in either gp45 or S3 (rev) were

obtained. Finally,EIAV apparentlyconsistsofamixture of

virusespresentatthe sametime.

Variationof the envelope gp9O glycoprotein and the

over-lapping S2 open reading frame. Rapid sequence variation

followedbyepitope-specific selection of EIAV variants able to escapeimmune surveillance have been implied as

impor-tant factors in the pathogenesis of persistent infection and

disease induced byEIAV (13, 32, 38). Ofthe 16 nucleotide

changeslocated inthe gp9O envelopeglycoprotein regionof

the Th-1 clones analyzed, 15 resulted in changes in the

predicted amino acid sequence, representing4.4%

variabil-ityattheamino acid level (Fig. 2). Furthermore,6of the 15

changes were amino acids potentially involved in

glycosyl-ation (asparagine, serine, and threonine). The changes at nucleotide 5762fromanalanine inMA-1 and the otherTh-1

clones to a threonine in Th-i clone Sall and atnucleotide 6233 fromanasparticacid inMA-1andthe otherTh-1clones

to an asparagine in Th-1 clone 7b41 actually did create

N-linked glycosylation sites. Interestingly, of the 15amino acid differences in the gp9Oregion, 4 are within the

hyper-variable region described by Payne et al. (48). If the area

includingtheflanking cysteinesisincluded(nucleotides 6176

to 6343), makingthis area intoa potential loopresembling

hypervariable loops seen in HIV (21, 35, 45, 48), 7 amino

acids arevariable, givinga heterogeneityashighas7 of56

(12.5%) amino acids. Interestingly, one of the amino acid

changesinvolvedoneofthemultiplecysteines flankingthis

area. Moreover, four of thechanges inthis regionincluded

amino acidspotentially involved inglycosylation, and one

did in factchange anN-linked glycosylationsite. Also, the

silent nucleotide change at position 6331 involved an

N-linkedglycosylation site. Thesefindings indicate, as

pre-6186 A (Lys) C(Thr*)

6195 T (Leu) C (Pro)

6221 G (Val) A (Ile)

6233 G (ASD) A(Asn*)

6284 A(Thr*) C(Pro)

6291 G(Ser*) T(Mle) T (MIe) T( le) T (Mle) T(Ile) T (MIe) T (Ile)

6331 C (Asn*) T (same)

6342 G (Cys) A(Tyr)

on November 10, 2019 by guest

http://jvi.asm.org/

gp45 reading frame in envelope

Clone

MA-I 8a5a 8a81 8a112

A(Thr*)

A(Asp)

A (Arg)

A(Arg)

A(Lys)

C (Arg)

G(Glu)

A (smue)

A (lie) A (tIe)

8a113 8a114

A(Thr)

A (Asp)

A(Arg)

G(Arg)

A (Arg)

A(Lys)

C(Arg) C (Arg)

G (Glu)

A (same) A (Ite) A(Ite)

C (Arg)

A (lLe)

7592 C (Gin) T (stop)

7596 A (Glu) G(Gly) G (Gly) G (Gly)

7617 G(Arg) A(Gin)

S3 open reading frame

Clones

NA-1 8a51 8a81 8112 8a113 8al14

NucLeotide

7319 G(Cys) A (Tyr) A (Tyr)

7380 G(Arg) A (same) A(same)

7382 G (Trp) A (stop) A (stop)

7395 A(Gin) G(sm)

7433 G (Trp) A(stop) A(stop)

7436 G (Arg) A(Lys) A (Lys)

T (Val)

A(Lys) G(Glu)

T (lIe)

C (Pro)

A (Arg)

G (Ser)

C (Ala)

G(Arg)

A(Lys)

A (Asn*)

C (Ala) C (Ala)

G (Arg)

A (Lys) A (Asn*) A(Asn*) A (Asn*)

T (Leu)

G (sam) G (sam)

A (sa)

FIG. 2-Continued.

C (Ala)

A(Asn*)

G(same)

viously suggested by Payne etal. (48, 49), thatvariation of

aminoacidsequenceandchanges in glycosylationpatternin

thisarea of the viral envelope mayplay important roles in

the development of antigenic andbiological variant viruses

and in the pathogenesis of persistent EIAV infection and chronic recurrent disease. Furthermore, our data suggest

that this variation can occur early in infection and can

alreadybedetectedasgenotypic variationin thefirstfebrile

cycle. Another area ofvariability was found in gp9O from

nucleotide 5743 to 5895, inwhich fouramino acid changes

werefound. Thisareacontainsoneofthe changed N-linked

glycosylation sites and has multiple cysteinesatthe

bound-aries, and it is possible that this area represents another

hypervariable loop. In contrast, the amino-terminal part of

gp9Oand theS2openreadingframewerehighlyconserved.

Although the function and significance of the S2 open

reading frameareundetermined, these resultsmayindicate

stringent structural and functional requirements for this

regionof thegenome.

Genetic variation has been shown to occur in HIV and

other lentivirus infections. This variation can occur both

withinanindividualover aperiod of time andas amixture of genotypes present atthesametime(4, 7, 9,10, 20, 21, 25, 36,

40, 55, 56, 64). The different genotypes present have been

shown to vary in biological phenotype, and it has been shown that this variation inpartisdeterminedbysequences

inthe viralenvelope (21, 30, 65). Virulence determinants of

immunosuppressive isolates of feline leukemia virus have

also beenmappedtonucleotidechangesin the viralenvelope

gene (47, 50), and env gene sequences were found to be

important fortheproductionofleukemiainducedbyFriend

and Rauscher murine leukemia viruses (34, 37, 54, 59).

Furthermore,thedifference betweenmacrophage-tropicand

T-cell-tropic HIVhasbeenmappedtotheenvelope,

partic-ularlysequences nearand in thehypervariable loop V3(21,

35). Notably, our isolates, Th-1 and MA-1, differ in their

abilitiestoreplicate inmacrophages (7) and have consider-ablevariation in thehypervariable region, perhaps indicating

asimilar celltropismdeterminantintheenvelopeof EIAV.

Also, it is important to note that the degree ofvariability

found in theTh-1isolate canbe detectedonlywhenisolates

are analyzed before repeated cell culture passage. It has

Nucleotide

7319

7380

7382

7395

7433

7436

7457

7463

7474

7481

G (Ala)

G(Gly)

G(Gly)

A (Lys)

G (Gly)

G(Glu)

T(Trp)

A (Lys)

G (Val)

T (Phe)

7457

7463

7474

7481

7592

7596

7617

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.134.475.76.492.2]

been shown for HIV and influenza virus that repeated passage will select for the fastest replicating virus without

thediverse pressure of in vivo biological and immunological

selection (21, 55). We PCR amplified and cloned our Th-1

EIAV after only one cell-free passage in HMC. Thus, our

clones should be representative of EIAV genotypes present

in vivo.

Analysis of the gp45 transmembrane glycoprotein. Of the13 nucleotide changes in the gp45 transmembrane protein re-gion analyzed, 12 resulted in amino acid changes (9.8% variability of the 123 amino acids), of which only one

involved potential glycosylation (Fig. 2). The biological

significance of most of the changes in this reading frame is at

present unclear. However, clone Th-18a81 had a stop codon

instead of a glutamine in this reading frame at nucleotide

7592, suggesting a premature termination of the gp45 at this

point. Interestingly, the stop codon in gp45 of clone Th-1 8a81 occurred in the same carboxy-terminal area of the

protein described for certain variants of EIAV passaged

repeatedly incultured equine kidney cells (52). This trunca-tion occurs close to the proposed cleavage site of gp45 which produces a 32-kDa glycosylated N-terminal segment and a

20-kDa nonglycosylated C-terminal fragment (52).

Trunca-tion at this point would suggest producTrunca-tion of only the

N-terminal segment. Furthermore, it has been shown that viruses having a truncated form of gp45 are more infectious

for cultured cells (52). In cell culture the stop codon was

changed from a glutamine or a glutamic acid codon, and

intriguingly, a stop codon changed from a glutamine codon

hasalsobeenreported in the transmembrane protein of a cell

culture-derived isolate of simian immunodeficiency virus

(31), exactly as reported here for EIAV in vivo. The

trun-cation of the simian immunodeficiency virus transmembrane protein only occurred when the virus was passaged in human

cells and not in monkey cells (31). A truncated transmem-brane protein for certain isolates of HIV types 1 and 2 has also recently been shown (22, 39). Our clones were derived from PCR-amplified DNA extracted from infected horse

monocyte/macrophage

cultures passaged only once. The

monocyte/macrophage lineage of cells is considered to be

the target cell for EIAV infection in vivo, and thus, our data

indicate thatlentiviruses having a truncated transmembrane

protein may play a role during in vivo infection. An exact

role of theC-terminal region of the lentivirus transmembrane

protein has not been defined; it is, however, tempting to speculate that this region is involved in viral replication or

cytopathogenicity in certain cells.

Analysis of theS3 (rev) open reading frame. In the S3 open

readingframe, overlapping the gp45 reading frame, only 7 of

the 13 nucleotide changes resulted in amino acid differences

and only 1 involved potential glycosylation. However, two

identical clones, Th-1 8a51 and 8a113, had two tryptophan

codonschanged to stop codons at nucleotides 7382 and 7433.

TheS3 open reading frame of EIAV is thought to encode a

lentivirustrans-acting regulatory protein termed rev (15, 51,

63). The rev protein of HIV and simian immunodeficiency

virus functions in the stability and transport of specific

incompletely spliced viral RNAs between the nucleus and

cytoplasm and isabsolutely required for expression of viral

structuralproteins. When functional rev is present,

cytoplas-mic RNAs consist primarily of unspliced genocytoplas-mic and

par-tially spliced env mRNAs. Without rev, cytoplasmic RNAs

consist ofmultiply spliced mRNAs (1, 18, 19, 26, 63).

Thus,

rev isimportant in the transition from early to late

transcrip-tion andreplication of thevirus (29). The stop codons in our

Th-1 clones both werechanged from tryptophan codons and

occurred early in the reading frame as described for in

vitro-derived rev-deficient phenotypes of EIAV (63). In the

twocellculture-derived clonespreviouslycharacterized, the

rev variants had either a 2-base insertion followed by a

tryptophan-to-stop codon change or a4-basedeletion.

Inter-estingly, this deletion occurred only 8 bases from the stop

codon in our Th-1 clones. It has been shown that cell

cultures infected with rev-deficient EIAV produce no

infec-tious virus and release few or noparticles from theinfected

cells. Furthermore, such infected cells contain

predomi-nantlymultiplyspliced RNA, and no full-lengthor envRNA

can be detected in the cytoplasm (63).

The Th-1 clones containing the rev stop codons also

contained an upstream nucleotide substitution atnucleotide

7319 which changed oneof the twocysteineresiduesin

S3

to

a tyrosine (Fig. 2). This is interesting considering that the

cysteine residues in retroviruses, including EIAV, are

usu-ally highly conserved (48). Taken together, these data

indi-cate that our clones represent sequences from EIAV

ge-nomes which are deficient in rev function (63) and that the

rev-deficient phenotype appears to relieve the pressure on

conservation of the twocysteines in this gene. Furthermore,

rev-deficient clones, whether obtained in vivo or in vitro,

seem to have a similar pattern of truncation, suggesting that

either an underlying basal mechanism for stop codon

intro-duction in this particular area, or, more interestingly, a

truncated

S3

open reading frame product, perhaps having

only one cysteine residue, may have a biological significant

function. rev-deficient viruses do not express viral proteins

at the cell surface and therefore can remain undetected by

the host immune system. Also, such infected cells do not

cause viral interference and can thus be reinfected with

replication-competent viruses. Therefore, rev-deficient

vi-ruses may play important roles in maintaining

persistent

infection and in recurrent disease. Until a comprehensive

and detailed transcriptional, translational, and functional

map including several isolates and phenotypes of EIAV is prepared, these intriguing questions cannot be answered.

Our data together with the emerging body of evidence from other lentivirus infections indicate that a heterogeneous population of EIAV genotypes may coexist in vivo and that genotypes of different antigenic makeup, cell tropism, cyto-pathogenicity, and replicative capabilities together initiate and sustain the development of persistent infection and chronic disease. Thus, it may be possible, perhaps by the development of pseudotypes or by protein complementa-tion, that the coexistence of replication-competent and -de-fective viruses infecting the same cell permits the amplifica-tion and transmission of the defective genotype, a scenario well known from avian and murine leukemia viruses and recently described for certain isolates of feline leukemia

virus (47).

ACKNOWLEDGMENTS

We thank Lene Arndrup Poulsen for excellent technical assis-tance.

This work was supported in part by grants to S.A. from the Danish Center for Animal Biotechnology Research, the Danish Agricultural and Veterinary Research Council, and the

Bernhard

Bang Foundation and by a grant to S.C. from the Iowa State University Agricultural Biotechnology Council.

REFERENCES

1. Ahmed, Y. F., S. M. Hanly, M. H. Malim, B. R.Cullen, and W. C. Greene. 1990. Structure-function analyses of the

HTLV-I

Rex and HIV-1 Rev RNA response elements: insights into the mechanism of Rex and Rev action. Genes Dev.4:1014-1022.

on November 10, 2019 by guest

http://jvi.asm.org/

2. Alexandersen, S., M. E. Bloom, and S. Perryman. 1988.Detailed transcription map of Aleutian mink disease parvovirus. J.Virol. 62:3684-3694.

3. Arya, S. K., C. Guo, S. F. Josephs, and F. Wong-Staal. 1985. trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69-73.

4. Asjo, B., L. Morfeldt-Manson, J. Albert, G. Biberfeld, A. Karisson, and K. Lidman. 1986. Replicative capacity of human immunodeficiency virus from patients withvaryingseverity of HIV infection. Lancetii:660-662.

5. Bloom, M. E., S. Alexandersen, S. Perryman, D. Lechner, and J. B. Wolfinbarger. 1988. Nucleotide sequence and genomic organization of Aleutian mink disease parvovirus (ADV): se-quence comparisons between anonpathogenicand apathogenic strain of ADV. J. Virol. 62:2903-2915.

6. Carpenter, S., S. Alexandersen, M. J. Long, S.Perryman,andB. Chesebro. 1991. Identification of ahypervariable region in the long terminal repeat of equineinfectiousanemiavirus. J. Virol. 65:1605-1610.

7. Carpenter, S., and B. Chesebro. 1989. Change in host cell tropism associated with in vitro replicationof equineinfectious anemia virus. J. Virol. 63:2492-2496.

8. Carpenter, S., L. H. Evans, M.Sevoian,and B.Chesebro.1987. Role of host immune responseinselectionofequineinfectious anemia virus variants. J. Virol.61:3783-3789.

9. Cheng-Mayer, C., D. Seto, M. Tateno, and J. A. Levy. 1988. Biologic features of HIV-1 that correlate withvirulence inthe host. Science240:80-82.

10. Chiodi, F., A. Valentin, B. Keys, S. Schwartz, B. Asjo, S. Gartner, M. Popovic, J. Albert, V.-A. Sundqvist, and E.-M. Fenyo. 1989. Biological characterization ofpaired human immu-nodeficiency virus type1isolates from blood and cerebrospinal fluid. Virology 173:178-187.

11. Chiu,I. M., A. Yaniv, J. E. Dahlberg, A. Gazit,S. F. Skuntz, S. R. Tronick,and S.A.Aaronson. 1985. Nucleotide sequence evidence for relationship of AIDS retrovirus to lentivirus. Nature (London)317:366-368.

12. Clements, J. E.,N.D'Antonio, and0.Narayan. 1982.Genomic changes associated with antigenic variation of visna virus. II. Commonnucleotidesequencechanges detected invariants from independent isolations. J.Mol. Biol. 158:415-434.

13. Clements,J. E.,S.L.Gdovin,R. C.Montelaro,and 0. Narayan. 1988. Antigenic variation in lentiviral diseases. Annu. Rev. Immunol. 6:139-159.

14. Derse, D., P. L. Dorn,L.Levy, R.M.Stephens, N. R. Rice, and J. W. Casey. 1987.Characterizationof equineinfectiousanemia virus long terminal repeat. J.Virol. 61:743-747.

15. Dorn,P., L. DaSilva,L.Martarano,and D. Derse. 1990. Equine infectious anemiavirustat:insights into the structure,function, and evolution of lentivirus trans-activator proteins. J. Virol. 64:1616-1624.

16. Duh, E.J.,W. J.Maury, T. M. Folks, A. S. Fauci, and A. B. Rabson. 1989. Tumornecrosis factor a activates human immu-nodeficiency virus type 1 through induction of nuclear factor binding to theNF-KB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA86:5974-5978.

17. Evans, K.S.,S.L.Carpenter, and M. Sevoian. 1984. Detection ofequine infectious anemia virus in horse leukocyte cultures derived fromhorsesinvariousstages ofequine infectius anemia viralinfection. Am. J. Vet. Res. 45:20-25.

18. Felber, B. K., C. M. Drysdale, and G. N. Pavlakis. 1990. Feedback regulationofhuman immunodeficiency virus type 1 expressionbythe Revprotein. J.Virol. 64:3734-3741. 19. Felber, B. K., M.Hadzopoulou-Cladaras,C. Cladaras, T.

Cope-land, and G. N. Pavlakis. 1989. revproteinofhuman immuno-deficiency virustype 1 affects the stability and transportofthe viralmRNA.Proc. Natl. Acad. Sci. USA 86:1495-1499. 20. Fenyo, E. M., L. Morfeldt-Manson, F. Chiodi,B. Lind,A. von

Gegerfelt, J. Albert, E. Olausson, and B.Asjo. 1988. Distinct replicative andcytopathic characteristics of human immunode-ficiencyvirus isolates. J. Virol. 62:4414 4419.

21. Fisher, A.G., B. Ensoli, D. Looney, A. Rose,R.C.Gallo,M.S. Saag, G. M. Shaw, B. H. Hahn, and F. Wong-Staal. 1988.

Biologically diverse molecular variants within a

single

HIV-1

isolate. Nature (London)334:444-47.

22. Franchini, G.,and M. L. Bosch.1989.Genetic relatedness ofthe

humanimmunodeficiencyviruses type1 and 2(HIV-1, HIV-2)

andthesimianimmunodeficiencyvirus(SIV).Ann. N.Y.Acad. Sci. 554:81-87.

23. Golub,E.I.,G.Li,and D. L.Volsky.1990. Differencesin basal

activity of the long terminal repeat determine the different

replicative capacitiesoftwoclosely related human

immunode-ficiencyvirustype 1isolates. J. Virol. 64:3654-3660.

24. Gonda,M. A., M. J. Braun, J. E. Clements,J. M.

Pyper,

F.

Wong-Staal,R.C.Gallo,and R. V.Gilden.1986.HumanT-cell

lymphotrophic virustype III shares sequence

homology

witha

family ofpathogenic lentiviruses. Proc. Natl. Acad. Sci. USA

83:4007-4011.

25. Hahn,B. H.,G. M. Shaw,M. E.Taylor, R. R.Redfield,P.D.

Markham, S. Z.Salahuddin,F. Wong-Staal,R. C.

Gallo,

E.S.

Parks,and W. P. Parks.1986.Genetic variation inHTLV/LAV overtime in patients with AIDS oratrisk for AIDS. Science 232:1548-1553.

26. Hanly,S.M.,L. T.Rimsky,M. H.Malim,J.H.Kim,J.Hauber,

M. D. Dodon,S.-Y. Le,J. V. Maizel,B. R. Cullen,andW. C. Greene. 1989.

Comparative

analysis

of the HTLV-IRex and HIV-1 Rev trans-regulatory

proteins

and theirRNA response elements. GenesDev. 3:1534-1544.

27. Issel, C. J., and L. Coggins. 1979.

Equine

infectious anemia:

currentknowledge.J. Am. Vet. Med. Assoc. 174:727-733.

28. Kawakami, T., L. Sherman, J. Dahlbert, A. Gazit, A. Yaniv,

S. R. Tronick,and S. A. Aaronson. 1987. Nucleotide sequence

analysis ofequine infectiousanemia

proviral

DNA.

Virology

158:300-312.

29. Kim, S., R. Byrn, J. Groopman, and D. Baltimore. 1989.

Temporal aspects ofDNA and RNA

synthesis during

human

immunodeficiencyvirusinfection: evidence for differentialgene

expression. J.Virol. 63:3708-3713.

30. Kim, S., K. Ikeuchi, J. Groopman, and D. Baltimore. 1990. Factorsaffectingcellular

tropism

ofhuman

immunodeficiency

virus.J. Virol.64:5600-5604.

31. Kodama, T., D. P. Wooley, Y. M. Naidu, H. W. Kestler III,

M.D.Daniel,Y.Li,and R.C. Desrosiers. 1989.

Significance

of

premature stop codons in env of simian

immunodeficiency

virus.J. Virol. 63:4709-4714.

32. Kono,Y.,K.Kobayashi,and Y.

Fukunaga.

1973.

Antigenic

drift

ofequineinfectiousanemiavirusin

chronically

infectedhorses. Arch. GesamteVirusforsch. 41:1-10.

33. Li,Y.,E.Golemis,J.W.

Hartley,

and N.

Hopkins.

1987. Disease

specificity ofnondefective Friend and

Moloney

murine leuke-mia viruses iscontrolled

by

asmall number of nucleotides. J.

Virol.61:693-700.

34. Linemeyer,D.L.,J.G.Menke,S. K.Ruscetti,L.H.Evans,and

E. M. Scolnick. 1982.

Envelope

gene sequences which encode thegpS2proteinof

spleen

focus-forming

virus are

required

for

the induction of

erythroid

cell

proliferation.

J. Virol. 43:223-233.

35. Liu, Z. Q., C. Wood, J. A. Levy, and C.

Cheng-Mayer.

1990.

Theviralenvelopegeneisinvolved in

macrophage tropism

ofa

human

immunodeficiency

virustype1strain isolatedfrombrain tissue.J. Virol.64:6148-6153.

36. Lutley,R.,G.Petursson,P. A.Palsson, G.

Georgsson,

J.Klein,

andN.Nathansson. 1983.

Antigenic

drift in visna: virus variation

during long-term infection of Icelandic

sheep.

J. Gen. Virol. 64:1433-1440.

37. Machida,C. A.,R. K.Bestwick,andD. Kabat. 1985. Aweakly

pathogenic Rauscher

spleen focus-forming

virus mutant that lacks the

carboxyl-terminal

membrane anchor ofits

envelope

glycoprotein. J. Virol. 55:990-993.

38. McGuire, T. C., K. O'Rourke, and W. P. Cheevers. 1987. A

reviewof

antigenic

variation

by

equine

infectious anemiavirus. Contrib. Microbiol. Immunol.8:77-89.

39. McNearney,T.,P.Westervelt,B.J.Thielan, D.B. Trowbridge,

J.Garcia, R.Whittier,andL. Ratner. 1990. Limited sequence

heterogeneity among

biologically

distinct human

immunodefi-ciency virus type 1 isolates from individuals involved in a

on November 10, 2019 by guest

http://jvi.asm.org/

clustered infectious outbreak. Proc. Natl. Acad. Sci. USA 87:1917-1921.

40. Meyerhans, A., R. Cheynier, J. Albert, M. Seth, J. Kwok, L. Sninsky, L. Morfeldt-Manson, B. Asjo, and S. Wain-Hobson. 1989.Temporalfluctuationsin HIVquasispeciesin vivo are not reflected bysequential HIV isolation. Cell 58:901-910. 41. Montagnier,L., C. Dauguet, C. Axler,S.Chamaret, J. Gruest,

M. T.Nugeyre,R. Rey, F.Barr*-Sinoussi, and J. C. Chermann. 1984. A new typeof retrovirus isolated frompatientspresenting with lymphadenopathy and acquired immune deficiency syn-drome: structural andantigenic relatedness with equine infec-tiousanemiavirus. Ann. Virol. 135E:119-134.

42. Montelaro, R.,J. Ball, P. Rwambo, and C. Issel. 1990. Antigenic variationduring persistent lentivirus infectionsand its implica-tions for vaccinedevelopment. Adv. Exp. Med.Biol. 251:251-272.

43. Montelaro, R. C., W. G. Robey, M. D. West, C. J. Issel, and P.J. Fischinger. 1988.Characterization oftheserological cross-reactivity between glycoproteins of the human immunodefi-ciencyvirusandequineinfectious anemia virus. J. Gen. Virol. 69:1711-1717.

44. Narayan, O., D. E. Griffin, and J. Chase. 1977.Antigenic shift of visnavirus inpersistently infected sheep. Science197:376-378. 45. Neurath, A.R., and N. Strick. 1990.Confronting the hypervari-ability ofanimmunodominant epitope eliciting virus neutraliz-ing antibodies from the envelope glycoprotein of the human immunodeficiency virustype 1(HIV-1). Mol. Immunol. 27:539-549.

46. Osborn, L., S. Kunkel, and G. J. Nabel. 1989. Tumornecrosis factor a and interleukin 1 stimulate the human immunodefi-ciency virus enhancer by activation of the nuclear factor KB. Proc. Natl.Acad. Sci. USA 86:2336-2340.

47. Overbaugh, J., P. R. Donahue, S. L. Quackenbush, E. A. Hoover, and J. I.Mullins. 1988. Molecularcloning ofafeline leukemia virus that induces fatal immunodeficiencydisease in cats. Science239:906-910.

48. Payne, S. L., F.-D. Fang, C.-P. Liu, B. R. Dhruva, P.Rwambo, C. J. Issel,and R. C. Montelaro. 1987. Antigenic variationand lentivirus persistence: variations in envelope gene sequences duringEIAVinfectionresemblechanges reportedforsequential isolatesof HIV.Virology 161:321-331.

49. Payne, S. L., K. Rushlow, B. R. Dhruva, C. J. Issel, and R. C. Montelaro. 1989. Localization of conserved and variable anti-genic domains of equine infectious anemia virus envelope glycoproteins usingrecombinantenv-encoded protein fragments produced inEscherichiacoli. Virology 172:609-615.

50. Poss, M. L., J. I. Mullins, and E. A. Hoover. 1989. Posttransla-tionalmodifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing felineleukemia virus retro-virus. J. Virol. 63:189-195.

51. Rasty,S., B. R. Dhruva, R. L. Schlitz,D. S. Shih, C. J. Issel, and R.C. Montelaro. 1990.Proviral DNA integration and transcrip-tionpatterns of equine infectious anemia virusduring persistent andcytopathic infections. J.Virol.64:86-95.

52. Rice, N. R., L. E. Henderson, R. C.Swoder, T. D. Copeland, S. Oroszlan,andJ.F.Edwards. 1990.Synthesisandprocessing of

the transmembrane envelope protein of equine infectious ane-mia virus. J. Virol. 64:3770-3778.

53. Rushlow, K., K. Olsen, G. Steigler, S. L. Payne, R. C. Mon-telaro, and C.J. Issel. 1986. Lentivirus genome organization: the complete nucleotide sequence of the env gene region ofequine infectious anemia virus. Virology155:309-321.

54. Ruta, M., R.Bestwick, C.Machida, and D. Kabat. 1983. Loss of leukemogenicity caused by mutations in the membrane glyco-protein structural gene of Friend spleen focus-forming virus. Proc.Natl. Acad. Sci. USA 80:4704-4708.

55. Saag, M. S., B. H. Hahn, J. Gibbons, Y. Li, E. S. Parks, W. P. Parks, and G. M. Shaw. 1988. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature (London) 334: 440-444.

56. Sakai, K., S. Dewhurst, X. Ma, and D. J. Volsky. 1988. Differ-ences incytopathogenicity and host cell range among infectious molecular clones of human immunodeficiency virus type 1 simultaneously isolated from an individual. J. Virol. 62:4078-4085.

57. Sanger, F., S.Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

58. Short, M. K., S. A. Okenquist, and J. Lenz. 1987. Correlation of leukemogenic potential of murine retroviruses with transcrip-tional tissue preference of the viral long terminal repeat. J. Virol. 61:1067-1072.

59. Sitbon, M., B. Sola, L. Evans, J. Nishio, S. F. Hayes, K. Nathanson, C. F. Garon, and B. Chesebro. 1986. Hemolytic anemia anderythroleukemia, two distinct pathogenic effects of Friend MuLV:mapping of the effects to different regions of the viralgenome.Cell47:851-859.

60. Sodroski, J., R. Patarca, C. A. Rosen, F.Wong-Staal,and W. A. Haseltine. 1985. Location of the trans-acting region on the genome of human T-cell lymphotropic virus type III. Science 229:74-77.

61. Sodroski,J., C. A. Rosen, F.Wong-Staal, S. Z. Salahuddin, M. Popovic, S. Arya, R. C. Gallo, and W. A. Haseltine. 1985. Transacting transcriptional regulation of human T-cellleukemia virustype IIIlongterminalrepeat. Science227:171-173. 62. Stephens, R. M., J. W. Casey, and N. R. Rice. 1986. Equine

infectious anemiavirusgag andpol genes: relatedness tovisna andAIDS virus. Science 231:589-594.

63. Stephens, R.M., D. Derse, and N. R. Rice. 1990. Cloningand characterization of cDNAs encoding equine infectious anemia virusTat andputative Revproteins.J. Virol.64:3716-3725. 64. Tersmette, M., R. A. Gruters, F. DeWolf, R. W. Y. DeGoede,

J. M. A. Lange, P. T. A. Scheliekens, J. Goudsmit, H. G. Huisman, and F. Miedema. 1989.Evidence forarole ofvirulent humanimmunodeficiency virus (HIV)variants in the pathogen-esis of acquired immunodeficiency syndrome: studies on se-quentialHIVisolates. J. Virol.63:2118-2125.

65. York-Higgins, D., C. Cheng-Mayer, D. Bauer, J. A. Levy, and D. Dina.1990.Humanimmunodeficiency virustype 1cellular host range,replication, andcytopathicityarelinked to theenvelope regionof the viral genome. J. Virol.64:4016-4020.

on November 10, 2019 by guest

http://jvi.asm.org/