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March 31,1992

DETECTION OF ANTIBODIES TO OVINE LENTIVIRUS USING A RECOMBINANT ANTIGEN DERIVED FROM THE env GENRE+ J. Kwang 1 . and R. C u t l i p 2

68933

IUSDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 2USDA, ARS, NADC, Ames, IA

50010

Received February i, 1992

SUbfl~ARY: The Western blot assay was performed to characterize antibodies to the transmembrane glycoprotein (TGP) of ovine progressive pneumonia virus (OPPV) by using glutathioue-S-transferase-TGP (GST-TGP) protein. The GST-TGP protein was efficiently expressed in Escherichia coli (E. coil) and was highly immunoreactive in the Western blot assay. This assay detected antibodies in 97% (103/106) of the sera from agarose gel immunodiffusion (AGID) positive OPP animals. Like human AIDS patients, antibodies to TGP appear to be one of the major serological markers in OPP infected animals.

Ovine progressive pneumonia, also known a s m a e d i and visna in Europe and Iceland, respectively, is caused by a nononcogenlc, exogenous retrovirus of the lentiviridae subfamily (1,2,3).

The disease is

characterized by a long latent period with an insidious onset and slowly progressive clinical course (4,5).

Consequently, the infection becomes

prevalent in a sheep population before it is recognized. The structure, genomic organization, and encoding sequences for the major structural proteins of visna virus strain 1514 have been established by determination of the complete nucleotlde sequence (6,7).

The ovine

lentivirus genome contains at least five genes (gag, pol, Q, tat, and env) and its overall organization is 5'-gag-pol-Q-tat-env-3' (6,7).

The mature

viral envelope glycoproteln gp135 is supposedly cleaved to produce an exterior N-termlnal glycoprotein and C-terminal transmembrane protein

+Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may be suitable. *To whom correspondence should be addressed. Abbreviations: TSP = transmembrane glycoproteln, AGID = agarose gel immunodiffuslon, OPPV = ovlne progressive pneumonia virus.

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(6).

The exterior and transmembrane proteins are approximately 556 and

327 amino acids, respectively (6). In human AIDS virus, the 160 kDa glycoproteln appears to be the precursor for the gpl20 (exterior) and gp41 (transmembrane) envelope glycoprotelns (8).

It has been shown that gp41 glycoproteln appears to be

one of the major serological markers by immunoblot analysis in human AIDS patients (9,10).

Although viral proteins of 14, 16, 25, 40, 70, 90, 105,

and 135 kDa have been detected in goat and sheep sera to caprine and ovine lentiviruses (11,12,13,14,15,16,17), the 40 kDa protein has not been characterized.

This 40 kDa protein is probably equivalent to the gp41

transmembrane glycoprotein of Human Immunodeficiency Virus (HIV).

In this

report, we describe the use of the polymerase chain reaction (PCR) to amplify a portion of the putative TGP gene from cloned Icelandic visna virus strain 1514 DNA.

The PCR product was cloned and expressed as a

fusion protein in E. coll.

This partially purified recombinant fusion

protein was highly immunoreactive in the immunoblot assay.

The assay

detected antibodies in 97% (103/106) of the sera from AGID positive OPP animals.

MATERIALS AND METHODS Serum samples. A panel of 113 sera were Included in this study. First, 106 samples were from experimentally or naturally infected sheep and had been characterized as OPP antibody positive by AGID test (5). These sera represent beginning, early, late, or unknown stages of infection. Second, six animal sera were used as the negative reference. These sera tested negative by AGID. Their peripheral blood lymphocytes tested negative for OPP provlral DNA by PCR amplification using the primers specific for the gag and pol gene regions. Third, one high titer OPP serum was used as positive reference. Bacterial strain and plasmids. Escherichia coli JMI05 was the host strain for all the recombinant plasmids. Procedures for purifying DNA, DNA manipulations, and bacterial transformations have been previously described (18). The plasmid pGex2T (Pharmacia, Piscataway, NJ) was used as starting material for constructing an expression vector. The pGex2T vector contains the glutathlone-S-transferase (GST) gene under the control of Tac promoter. SDS-p01yacrylamide gel el ectrophoresis and Western blottin_~g. These procedures were performed by the standard protocols (19,20). Purification of GST-TGP polypeptide. The procedure for inducing expression and preparation of bacterial lysates for gel analysis has been described in detail previously (21). For purification of fusion protein, the bacterial cell pellets were suspended in 50 mM Trls-HCl (pH 8.0) - 0.5 mM EDTA 0.3 M NaCI - 1 mg/ml lysozyme - 0.5% Nonidet p40. The cells were sonicated and centrifuged at 15,000 x g for i0 min. The pellet was then extracted with 2 M urea, 8 M urea buffer (both in 25 mM Tris [pH 8.0] i0 mM EDTA), and SDS buffer (1.5% SDS, 50 mM dithiothreltal) sequentially. Construction of the pGex2T-OPP-TGP expression plasmid, pLVI-IKSI plasmid containing a complete infectious vlsna provlral DNA was kindly -

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

provided by Dr. Katherine Staskus (22). In order to select an appropriate gene fragment for expression, PCR reactions were carried out using pLVI-IKSI as a template. The primers (5'- AA GGA TCC ATG GTA TAC CAA GAA TTG -3' and 3'- ACC AAG AGC ACC GAG TTT CTT AAG GT -5') were used to amplify the 282 bp transmembrane region of the env gene. The amplified sequence includes nucleotides 8186 to 8467, according to the published sequence (22). The BamH I and EcoR I restriction sites were incorporated at the 5' ends of the primers to facilitate cloning. The primers and amplified sequences were designed to be in frame with GST of pGex2T expression vector. The amplified PCR product was of the expected length, 282 bp, and was digested with BamH I and EcoR I. It was then subjected to electrophoresis in 1.5% low melting temperature agarose gel. The 282 bp BamH I/EcoR I band was isolated and inserted between the BamH I and EcoR I sites of the pGex2T plasmid to create in-frame fusion with GST protein. The resulting plasmid--pGex2T-OVL-TGP--is shown in Figure I.

RESULTS AND DISCUSSION Expression of GST-TGP fusion polypeptide in E. coll.

Nucleotides

8186 to 8467 of the ovine lentivirus DNA (Icelandic strain) was amplified through PCR techniques,

incorporating a BamH I linker at the 5' end and an

EcoR I linker at the 3' end.

The BamH I-EcoR I fragment was fused in

sequence to encode the GST protein under control of the Tac promoter (Fig. i).

Hydrophilicity and hydrophobicity analyses of the amino acid sequence

indicated that this portion of TGP was hydrophilic and most likely to be on the surface of the molecule.

The calculated molecular weight of this

fragment is 11.5 kDa. When the pGex2T-OPP-TGP plasmid was introduced in E. coli JMI05 host cell and induced by isopropyl-B-D-thiogalactopyranoside

(IPTG), the

construct was found to express high levels of a 37.5 kDa fusion protein on Coomassie blue stained gels, consistent with the predicted size (GST 26 kDa plus OPP TGP 11.5 kDa) (Fig. 2, Lane 4).

This protein was not

observed in uninduced cells containing pGex2T-OVL-TGP or induced cells containing the pGex2T vector without an insert (Fig. 2, Lanes 2 & 3). Sequential extraction of the insoluble fraction of the induced bacterial cells with 8 M urea and SDS buffer were enriched for GST-TGP (Fig. 2, Lanes 5 & 6).

Although a small amount of GST-TGP was left in 8 M urea

buffer, most of it was recovered in the SDS buffer (Fig. 2, Lanes 5 & 6). Characterization of GST-TGP polypeptide.

To characterize the 37.5 kDa

recombinant protein, the protein was tested for immunogenicity by (i) reacting with high titered sheep anti-OPP serum (positive control serum) and (ii) eliminating the possibility that the antisera recognized the GST moiety and not the TGP moiety.

Bacterial cell extracts containing the

GST-TGP and GST only were tested by Western blot.

Prominent bands at 37.5

kDa were seen in Western blots of GST-TGP when reacted with high titer

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TrpPhe Ser Trp Leu Lys TGG TIC TCG TGG CTC AAA ACC hAG AGC ACC GAG "i"1-I"C'I-FhAG 5'

BamHI Met Val Tyr Gin Glu Leu 5' GGA TCC ATG GTA TAC CAA GAA l"rG - - - - J . ~ TAC CAT ATG G'I-r G'l-l" AAC

I

I E~oR,

8186

8467

Fig. I. Schematic representation of the pGex2T-OVL-TGP expression plasmid. The amplified sequence from 8186 to 8467, encoding 94 amino acids of the ovine lentivirus TGP, was inserted into the pGex2T expression vector. Resulting was the expression of a 37.5 kDa fusion protein. Abbreviations: B, BamH I; E, EcoR I; TGP, transmembrane glycoprotein; and GST, glutathione-S-transferase.

sheep serum (Fig. 3A, Lane I), but not in those reacted with serum from a negative

control sheep (Fig. 3A, Lane 2).

Neither positive nor negative

sheep reacted with the GST control (Fig. 3B, Lanes

i & 2).

A guinea pig

antiserum to purified GST identified a 37.5 kDa band on the Western blot of GST-TGP and 26 kDa band on the GST blot (Figs. 3A & 3B, Lane 3). data shown here indicates properties

The

that 37.5 kDa protein has the expected

of the GST-TGP fusion protein.

Western blots were due to cross-reaction

The extra bands found on of sera, probably due to

infection of the animals with E. coll. Reactions

of antibodies

GST-TGP provided sufficient selected sheep sera.

in OPP infected sheep.

antigen for seropositivity

survey among 112

These sera were first screened with AGID:

were OPP positive and six were negative to determine

Partially purified

the proportion

(Materials and Methods).

106 sera In order

of AGID positive individuals with antibodies

to TGP, all sera and a positive control serum were subjected to a Western blot assay. (103/106)

When GST-TGP was employed

of AGID positive sera demonstrated

(3/106) went undetected.

antibodies

Among the positives,

strong reactive band and the remaining assay.

in a Western blotting analysis,

These weakly positive sera were identified

infected sheep having an early stage of infection,

1043

to TGP and 3%

87% (90/103) produced a

13% (13/103)

the first time turning OPP positive by AGID.

97%

reacted weakly in this from experimentally and were recorded for

None of the six negative

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3

4

5

6

A

B

123

123

43KGST-TGP

29K-

®

II

GST

Fig.2. SDS-PAGE analysis of recombinant protein GST-TGP with Coomassie blue R-250 stain. Lane I Molecular standards in kilodaltons Lane 2 Induced cell containing the pGex2T vector Lane 3 Uninduced cell containing the pGex2T-OVL-TGP Lane 4 Induced cell containing the pGex2T-0VL-TGP Lane 5 8 M urea extraction fraction Lane 6 SDS extraction fraction GST-TGP recombinant protein is indicated by an arrow. GST is indicated by an asterisk. Fig~ 3. Western blot analysis of GST-TGP (A) and GST (B) proteins were electrophoresed and electroblotted onto nitrocellulose paper. Western blot strips were reacted with: I) Positive sheep sera 2) Negative sheep sara 3) Guinea pig anti-GST serum

control sera recognized immunogenic

the TGP.

Thus,

the recombinant

and is recognized by most individual

lentivirus.

Representative

protein is highly

sheep infected with ovine

Western blots are shown in Figure 4.

In studying the immune response to individual

ovine lentiviral

proteins by Western blot, Houwers and Nauta (16) found that antibodies p25 appeared first after infection and antibodies later.

These findings and our results suggested

sera samples were in the beginning insufficient investigate

antibody production

shown).

emerged

that these three negative

stages of infection and had

to TGP at this stage.

the reason for the discrepancy

tested positive in the AGID but negative expressed

to glycoproteins

to

To further

between the three sara that

in the TGP Western blot assay, we

the full length of p25 with GST fusion protein (data not This recombinant

monoclonal

protein (GST-p25) was recognized by the

antibody to OPPV-p25

(23) (Fig. 5).

the Western blot assay and, as expected, positively with p25 (Fig. 5). during the very beginning

GST-p25 was then used in

those three sera did react

Our preliminary

results

stage of OPP infection,

enough TGP antibody for detection,

indicated that

sheep may not produce

which is consistent with Houwers and

Nauta's data (16). Since antibodies OPP-infected development

to TGP were found in most of the examined sara of

sheep, TGP may serve as an important of an enzyme-linked

immunoabsorbant

epitope for the

assay (ELISA).

Currently,

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3

4

5

6

7 89

12

NN

34

N N

@

5

-gst-p25

.......

iNN ~

®

Fig. 4. Detection of recombinant GST-TGP by Western blot analysis. A total of ll3 sheep sera have been subjected into this analysis and the following representative blot was shown. The arrow indicates the position of GST-TGP. Lane I Negative control serum Lane 2 Positive control serum Lanes 3, 4, 5, & 6 Strong reactive serum Lanes 7 & 8 Weakly reactive serum Lane 9 AGID positive, Western negative serum ~ . Western blot analysis of GST-p25. Lane 1 Negative control serum Lanes 2, 3, & 4 AGID positive, GST-TGP Western blot negative serum Lane 5 Monoclonal antibody to OPP-p25. Lower bands were the breakdown products.

we are working on an oligopolypeptide enzyme immunoassay.

Such an assay

may offer a rapid, inexpensive, and sensitive method of detection as an option to the more expensive Western blot.

ACKNOWLEDGMENTS We thank Dr. Katherine Staskus for providing the pLVI-IKSI plasmid and her critical review of the manuscript, Dr. L. D. Pearson for providing the anti-OPP-p25 monoclonal antibody, Dr. E. T. Littledike for helpful discussions, Nancy Ferrell for excellent technical contributions, Joan Rosch for careful preparation of the manuscript, and Penny Bures for photographic assistance.

REFERENCES i. 2. 3. 4. 5. 6.

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Braun, M.J., Clements, J.E., and Gonda, M.A. (1987) J. Virol. 61, 4046-4054. Sarngadharan, M.G., Popovic, M., Bruch, L., Schupbach, J., and Gallo, R.C. (1984) Science 224, 506-508. Veronese Di Marzo, F., DeVico, A.L., Copeland, T.D., 0roszlan, S., Gallo, R.C., and Sarngadharan, M.G. (1985) Science 229, 1402-1405. Gnann, J.W., Nelson, J.A., and Oldstone, M.B.A. (1987) J. Virol. 61, 2639-2641. Bruns, M., and Frenzel, B. (1979) Virology 97, 207-211. Gogolewskl, R.P., Adams, D.S., McGuire, T.C., Banks, K.L., and Cheevers, W.P. (1985) J. Gen. Virol. 66, 1233-1240. Klein, J.R., Martin, J., Grlffing, S., Nathanson, N., Gorham, J., Shen, D.T., Petursson, G., Georgsson, G., Pausson, P.A., and Lutley, R. (1985) Res. Vet. Scl, 38, 129-133. Adams, D.S., and C~rham, J.R. (1986) Res. Vet. Sci. 40, 157-160. Stanley, J., Bhadurl, L.M., Narayan, O., and Clements, J.E. (1987) J. Virol. 61, 1019-1028. Houwers, D.J., and Nauta, I.M. (1989) Vet. Microb. 19, 127-139. Zanonl, R., Krlef, A., and Peterhans, E. (1989) J. Clin. Microb. 27, 580-582. Manlatls, T., Fritsch, E.F., and Sambrook, J. (1982) In Molecular ~ : A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Laemmll, U. (1970) Nature (London) 227, 680-685. Burnett, W.N. (1981) Anal. Biochem. 112, 195-203. Kwang, J., Bolin, S., Littledike, E.T., Dubovi, E.J., and Donis, R.O. (1991) Biochem. Biophy. Res. Comm. 178, 1326-1334. Staskus, K.A., Retzel, E.F., Lewis, E.D., Silsby, J.L., Cyr, S.S., Rank, J.M., Wietgrefe, S.W., Haase, A.T., Cook, R., Fast, D., Geiser, P.T., Harty, J.T., Kong, S.H., Lahti, C.J., Neufeld, T.P., Porter, T.E., Shoop, E., and Zachow, K.R. (1991) Virology 181, 228-240. Marom, K.A., Pearson, L.D., Chung, C.S., Poulson, J.M., and DeMartini, J.C. (1991) J. Clin. Microb. 29,1472-1479.

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