Vol. 64, No. 4

JOURNAL OF VIROLOGY, Apr. 1990, p. 1429-1436

0022-538X/90/041429-08$02.00/0 Copyright C 1990, American Society for Microbiology

Biological Activities of a Synthetic Peptide Composed of Two Unlinked Domains from a Retroviral Transmembrane Protein Sequence DONALD E. WEGEMER, KAREN G. KABAT, AND WILLIAM S. KLOETZER*

The R. W. Johnson Pharmaceutical Research Institute, San Diego, California 92121 Received 29 August 1989/Accepted 12 December 1989

We report several biological activities of a synthetic peptide whose sequence contains the highly conserved region of feline leukemia virus transmembrane protein (TM) synthetically linked to another short TM-derived sequence particularly rich in polar positive residues. This 29-amino-acid peptide blocked [3H]thymidine uptake 30 to 50% by concanavalin A-stimulated CD4+- but not CD8+-enriched murine splenocytes. Maximal suppression was detected at 12.5 ,ug (3 ,uM) to 75 ,ug (19 ,IM) per ml of growth medium; stimulation of [3H]thymidine uptake was observed at higher peptide concentrations. The synthetic peptide inhibited but did not stimulate [3lHlthymidine uptake by mitogen-activated thymocytes and antibody production by splenocytes as determined in a liquid hemolytic plaque assay. Similarities are reported between a consensus sequence of diverse retroviral TMs and a region of alpha interferons shown by others to be important for antiviral and cytostatic properties. The TM sequence-derived synthetic peptide blocked in a nontoxic and sequence-specific manner the release of murine leukemia virus from two chronically infected cell lines. We suggest that some of the biological effects of retroviral TM are mediated through a common pathway shared with alpha interferons.

Persistent viremia of cats infected with feline leukemia virus (FeLV) causes a variety of degenerative hematopoietic disorders including nonregenerative anemia, panleukemialike syndrome, and immunosuppression (13). Some clinical signs of these diseases may be directly caused by the retroviral transmembrane protein (TM) (27). In vitro activities suppressed by purified viral TM include feline neutrophil activation (20), [3H]thymidine uptake by concanavalin Astimulated feline peripheral blood leukocytes (22), and colony-forming units-erythroid, but not colony-forming unitsgranulocyte/macrophage, formation by feline bone marrow cells (36). Purified TM also inhibits [3H]thymidine uptake by established murine cell lines including an interleukin-2 (IL2)-stimulated cytotoxic T-cell line (called CTLL-2) (28, 29) and an interleukin-1 (IL-1)-stimulated T-helper-cell line (called DlO.G4.1) (29). A recombinant DNA fragment of murine leukemia virus (MuLV) TM expressed as a fusion protein in Escherichia coli also blocks [3H]thymidine uptake by IL-2-stimulated CTLL-2 cells, IL-1-stimulated D1O.G4.1 cells, and anti-CD3 monoclonal antibody-stimulated human peripheral blood leukocytes (31). These results strongly support a concept that retroviral TM plays an important functional role in virus-induced diseases. Sequence comparisons of envelope precursor proteins from many type C and D retroviruses reveal a highly conserved domain within the TMs (3). A 17-amino-acid peptide synthesized from the highly conserved region of MuLV TM, when carbodiimide cross-linked to bovine serum albumin, displays in vitro properties similar to those of UV-inactivated virus and partially purified TM (2, 11, 17, 29). Based on the high degree of sequence conservation and the biological properties of this 17-amino-acid synthetic immunosuppressive peptide, abbreviated ISP in the current report, it is likely that the highly conserved sequence of retroviral TM plays an important role in mediating at least

*

some of the biological activities reported for the intact molecule. The synthetic ISP is inactive unless chemically crosslinked to bovine serum albumin (2). We report several biological activities of a peptide whose sequence contains the highly conserved ISP homolog of the FeLV envelope precursor synthetically linked to another short TM-derived sequence (abbreviated P+) particularly rich in polar positive residues. This 29-amino-acid peptide, referred to in this report as P+:ISP, displayed some but not all in vitro activities reported for albumin-linked peptide without crosslinking to a large stabilizing protein. We show that P+:ISP altered [3H]thymidine uptake by concanavalin A-stimulated murine thymocytes and CD4+- but not CD8+-enriched splenocytes. P :ISP also blocked hemolytic plaque formation by murine splenocytes primed in vivo with sheep erythrocytes (SRBC) and then boosted in vitro with SRBC in the presence of peptide. Unlike reports on albumin-linked peptide activities, we found no evidence for P+:ISP suppression of [3H]thymidine uptake by IL-lp-stimulated U373 cells (17), IL-2 stimulated CTLL-2 cells, or mixed lymphocyte reactions (2). The cellular mechanism of retroviral TM-mediated suppression has not yet been clearly identified. We report distant similarities between the highly conserved ISP sequence and a domain of alpha interferons shown by others (1, 21, 32) to be important for antiviral and cytostatic activities. The synthetic P+:ISP inhibited in a sequencespecific and nontoxic manner the release of MuLV from two chronically infected cell lines. On the basis of these observations, we suggest that retroviral TM expresses some of its antiviral and cytostatic effects through a pathway shared with alpha interferons.

MATERIALS AND METHODS Cells and reagents. Murine thymocytes and splenocytes were isolated from 8- to 12-week-old BALB/c or C57BL/6 mice. The clone 1 Moloney MuLV-infected NIH 3T3 cell

Corresponding author. 1429

1430

WEGEMER ET AL.

line (7) and Rauscher MuLV-infected JLS-V16 cell line (26) were used for assessing antiviral activity. Murine monoclonal antibody and Low-Tox-M rabbit complement (Accurate Chemical Corp., Westbury, N.Y.) were used to selectively lyse splenic cell populations. Anti-L3T4 monoclonal antibody was generously provided by Michael Bevan (Scripps Research Institute). Monoclonal antibodies against Thyl.2, Lytl.2, and Lyt2.2 (Accurate); concanavalin A (Sigma Chemical Co., St. Louis, Mo.); alpha-beta interferon (4 x 106 U/mg, 8 x 105 U/ml; Lee Biomolecular) and all radioisotopes were purchased from commercial sources. Polyclonal antiserum to a synthetic peptide glutaraldehyde crosslinked to the carrier protein keyhole limpet hemocyanin was generated in rabbits as previously described (18). Protein sequence analysis and peptide synthesis. Predictions of secondary structure (8), hydropathicity (15), antigenic index (16), and surface probability (6) were determined with the PEPTIDESTRUCTURE and PLOTSTRUCTURE programs in the Sequence Analysis Software Package, Version 5.0 (Genetics Computer Group). All other predictions were made by using programs in PC Gene, Version 6.01 (Intelligenetics, Inc.). Protein sequence alignments were performed with the PALIGN program (24). Sequence alignment of the ISP domain from FeLV TM with similar regions of murine alpha1, human alpha2, and human alpha7 interferons were performed with the structure-genetic matrix, an open gap cost of 4, and a unit gap cost of 3. Peptides were synthesized by automated solid-phase methods. A region of FeLV TM containing a cluster of polar positive residues (Ala-Lys-Leu-Arg-Glu-Arg-Leu-Lys-GlnArg-Gln-Gln), abbreviated P+, was synthetically linked to the amino terminus of the FeLV ISP sequence (Leu-Gln-

Asn-Arg-Arg-Gly-Leu-Asp-Ile-Leu-Phe-Leu-Gln-Glu-GlyGly-Leu) to form a sequence called P+:ISP. Variations of this peptide sequence include a reversed ISP sequence linked to the carboxyl terminus of the polar positive moiety (P+ :PSI) and the polar positive sequence linked to the carboxyl terminus of ISP (ISP:P+). A synthetic peptide (Lys-Glu-Lys-Glu-Arg-Asp-Arg-Arg-Arg-His-Arg-Glu-MetSer-Lys) from the Moloney MuLV capsid antigen (CA) sequence was used for antiserum production. Mitogen stimulation of [3H]thymidine uptake. BALB/c (1 x 105) splenocytes were incubated for 4 days in a total assay volume of 200 ,ul containing 1 ,ug of concanavalin A per ml of RPMI 1640 growth medium (supplemented with 5% fetal calf serum [heated 30 min at 56°C], L-glutamine, sodium pyruvate, 2-mercaptoethanol, and gentamicin). At the end of 3 days, 0.2 ,uCi of [methyl-3H]thymidine (74 GBq/mmol) was added to each culture and then incubated overnight. The cells were harvested onto glass fiber filters with an automated cell harvester (Cambridge Biotechnology); the immobilized cells were then washed and dried, and their radioactivity was counted in ScintiVerse E (Fisher Scientific Co., Pittsburgh, Pa.) scintillation fluid. Quadruplicate samples were routinely used in all experiments. Liquid hemolytic plaque assay for antibody production. C57BL/6 mice were intravenously injected in the tail vein with 0.2 ml of a 1% SRBC suspension in Hanks balanced salt solution. After 3 days, the spleens were removed and splenocytes were cultured for 3 days in the presence of SRBC and synthetic peptide. Viable splenocytes (1 x 103, 3 X 103, and 1 x 104) were then sedimented by- low-speed centrifugation in 96-well tissue culture plates (Costar, Cambridge, Mass.) previously coated with poly-L-lysine and SRBC. Guinea pig complement (GIBCO Laboratories, Grand Island, N.Y.) was added after a 1-h incubation at

J. VIROL.

37°C. Further incubation at 37°C yielded clearly detectable hemolytic plaques within 2 h. Immunoblot detection of cell- and virus-associated capsid antigen. MuLV-infected cells (105) were incubated for 1 day in the presence of murine alpha-beta interferon or synthetic peptide. The conditioned growth medium was removed and clarified of cell debris by low-speed centrifugation. Highspeed centrifugation (2 h at 60,000 x g) of the clarified medium was used to generate a virus-containing pellet. The cells were rinsed with phosphate-buffered saline before solubilization. Cells and virus were suspended and boiled in sodium dodecyl sulfate sample buffer. Proteins were separated on 12% resolving gels by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (19) and then electrophoretically transferred to nitrocellulose (35). Viral CA was sequentially labeled with rabbit antiserum to a synthetic peptide and 0.5 ,uCi of '25I-protein A (3.7 MBq/0.1 ml) per sample. Detection of virus-associated reverse transcriptase activity. MuLV-infected cells (5.0 x 104) were incubated for 1 day in the presence of murine alpha-beta interferon or synthetic peptide. Conditioned growth medium was clarified by lowspeed centrifugation, and virus was concentrated by precipitation with a final concentration of 10% (vol/vol) polyethylene glycol. The viral pellets were suspended in 25 ,ul of a detergent-containing buffer (50 mM Tris hydrochloride, 10% glycerol, 0.2% Triton X-100, 40 mM KCl, pH 8.2). After the addition of an equal volume of assay mixture [50 mM Tris hydrochloride, 8 mM dithiothreitol, 1 mM MnCl2, 16 ,uCi of

[methyl-3H]deoxythymidine 5'-triphosphate (3,071 GBq/ mmol), 0.016 U of poly(rA) poly(dT)12-18, pH 8.2], the suspension was incubated for 60 min at 37°C. The entire reaction mixture was absorbed to DEAE-cellulose paper (DE81; Whatman, Inc., Clifton, N.J.), washed extensively with 5% (wt/vol) Na2HPO4, and dried, and then its radioactivity was counted in scintillation fluid. RESULTS Prediction of surface-exposed domains on retroviral TM. Figure 1 shows a secondary structure prediction for the TM region of the envelope precursor from Gardner-Arnstein strain FeLV (5). Superimposed on the structural backbone are domains of high antigenic indices and surface probabilities. The antigenic index is an algorithm designed to predict surface domains from combined values of flexibility, hydropathy, and solvent accessibility (16). Three of the four regions with high antigenic indices lie within the ISP domain (residues 534 to 550). The fourth antigenic region is located near the carboxyl terminus, which is presumably intracellular-intraviral to a predicted transmembrane domain. The envelope region 573 to 584 contains a prominent cluster of polar positive residues which accounts for the high surface probability value. Although this highly polar positive region is not shown to have a high antigenic index, other methods of predicting antigenic sites (15) predicted this polar positive domain to be the most highly antigenic site along the entire TM sequence. Based on predictions of antigenic index and surface probability, we suggest that both the ISP (residues 534 to 550) and polar positive (residues 573 to 584) domains are exposed at the external face of the retroviral membrane. Synthetic attachment of polar positive tails has been shown by others to enhance the biological activity of oligopeptides. The sequence Arg-Gly-Asp-Val has a definite but somewhat low affinity for the fibrinogen receptor on thrombin-activated platelets. Synthetic linking of polar pos-

BIOLOGICAL ACTIVITIES OF TM-DERIVED SYNTHETIC PEPTIDE

VOL. 64, 1990

NH2

-A

1431

_

A b

A c

_'A a

_ _j ISP

* Antigen.Index > 1.2 O Surface Probability > 5.0

polar(+

H02C

_

FIG. 1. Secondary structure prediction of the TM portion (residues 466 to 645) of the envelope precursor protein from Gardner-Arnstein strain FeLV. a, Alpha helices; b, beta sheets; c, random coils; d, beta turns.

itive residues to the amino terminus of Arg-Gly-Asp-Val greatly enhanced the ability of the peptide to block native fibrinogen attachment to activated platelets (30). We surmised that charge effects might also contribute to the interaction of viral TM with a putative cell receptor and that attachment of a polar positive sequence selected from the envelope TM sequence (precursor residues 573 to 584) to ISP might also activate or enhance biological activity of ISP. Hybrid peptides alter [3H]thymidine uptake by mitogenactivated CD4+ T cells. Synthetic ISP from the MuLV TM sequence is not biologically active unless cross-linked to a large carrier protein (2). Similar results were observed with unlinked FeLV ISP and peptides composed only of the TM cluster of polar positive residues (abbreviated P+) (data not shown). Figure 2 shows the effects of three peptides on [3H]thymidine uptake by concanavalin A-stimulated murine splenocytes and thymocytes. The P :ISP maximally blocked activated splenocyte uptake of [3H]thymidine by 30 to 50% at peptide concentrations from 12.5 ,ug (3 ,uM) to 75 ,ug (19 ,uM) per ml of growth medium (Fig. 2A). At higher peptide concentrations, [3H]thymidine uptake was stimulated to levels higher than in the mitogen-activated cultures without synthetic peptide. A similar experiment with murine thymocytes showed some inhibition of [3H]thymidine uptake but only at peptide concentrations over 75 ,ug/ml of growth medium. No suppressive effects have yet been detected by P :ISP in phytohemagglutinin-stimulated cultures, lipopolysaccharide-stimulated cultures, or mixed lymphocyte reactions (data not shown). No differences in biological activity

were found between the linear monomeric P+ :ISP and purified dimeric or cyclic forms created by the addition of amino- and/or carboxyl-terminal cysteine residues (data not

shown). Figure 2B shows the results of a similar comparison with a peptide composed of the same polar positive sequence linked to the amino terminus of a reversed ISP sequence (P+:PSI). The P+:PSI showed no inhibitory or stimulatory effects on either activated splenocytes or thymocytes. Figure 2C shows the results of still another comparison with a peptide composed of the same polar positive sequence linked to the carboxyl terminus of the ISP sequence (ISP: P+). The ISP:P+ activity on splenocytes was less suppressive than that of P+:ISP at low peptide concentrations and had no stimulatory effect at higher concentrations. The functional cell populations required for the P+:ISP effects on [3H]thymidine uptake were examined by cell depletion experiments with monoclonal antibody-mediated cytolysis before mitogen and peptide exposure. Figure 3A shows that splenocyte depletion of cytotoxic T cells with anti-Lyt2.2 monoclonal antibody had little qualitative effect on activated cell response to P+:ISP. Splenocyte depletion of T helper-inducer cells with either anti-L3T4 or anti-Lytl.2 monoclonal antibody eliminated a cell population essential for the observed alterations in [3H]thymidine uptake. Control experiments not shown included the culturing of mitogen-activated splenocytes in the presence of peptide and monoclonal antibody without complement, which showed no effect on peptide-mediated suppression. A second control

WEGEMER ET AL.

1432

J. VIROL.

experiment demonstrated that antibody had no mitogenic effect on splenocyte uptake of [3H]thymidine. Splenocytes responsive to P+:ISP did not adhere to nylon wool (data not shown). Figure 3B shows the effect P+:ISP had on mitogen-stimulated cells depleted first by passage through a nylon wool column and subsequently lysed with monoclonal antibodies and complement. Cytolysis of the nylon wool column-depleted Lyt2.2+ cells had little effect on the response to peptide. Cytolysis of Lytl.2+ cells again eliminated a cell population essential for P+:ISP responsiveness. Similar depletion experiments with monoclonal antibody to Mac-I antigen showed no qualitative effect on [3H]thymidine uptake, which probably excludes macrophages as direct mediators of the observed PJ:ISP effects (data not shown). The inhibitory-stimulatory effects of P+: ISP appear to be specific for splenic CD4+ T cells. This

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Peptide Cfonc (g/m) FIG. 2. Synthetic peptides alter [3H]thymidine uptake by concanavalin A-stimulated splenocytes and thymocytes. Stimulation index (SI) was defined as experiment;al counts per minute/counts per minute of unstimulated cells with out peptide. These results are typical of five or more experiment-s. (A) P+:ISP; (B) P+:PSI; (C)

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play a role in the T-cell response to P+:ISP. P+:ISP inhibits hemolytic plaque formation. Spleens from C57BL/6 mice primed with SRBC were removed, the splenocytes were isolated, and cell cultures were incubated for 3 days in the presence of SRBC with either P+ :ISP or P :PSI. After the 3-day incubation, anti-SRBC antibody levels were determined by briefly culturing the activated and immobilized splenocytes with complement and SRBC. The results shown in Fig. 4 indicate that P+ ISP, but not P :PSI, specifically inhibited the T-helper-cell-dependent activation of B cells in response to antigen and the resulting formation of hemolytic plaques. Sequential relatedness of retroviral TM and alpha interferons. Comparison of all retroviral envelope sequences in the Swiss Protein Databank (Version 11) showed that 20 envelope precursor proteins contain the consensus sequence Gln-Asn-Arg-Arg-Gly-Leu-Asp-Xnn-Leu. One conceivable way that TM-related proteins might exert their effects is through a cytokine receptor. We compared the TM consensus sequence with all entries in the Protein Databank and found distant similarities to a region of the human alpha7 interferon sequence. Figure 5 shows an alignment between portions of the FeLV TM and three alpha interferon sequences. Precursor protein regions corresponding to residues 33 to 67 of human alpha7 interferon (21), residues 28 to 38 of human alpha2 interferon (32), and residues 33 to 43 of murine alpha1 interferon (1) have been shown by others to be important for antiviral and cytostatic activities. Antiviral effects of P+:ISP on retrovirus-infected cell lines. Because of the similarities between the TM consensus sequence and the functionally important domain of three alpha interferons, we decided to test the effects of P+ :ISP on retrovirus production by chronically infected cell lines. Antiviral activity was assessed by two methods: the release of virus-associated reverse transcriptase activity and CA expression as detected by immunoblotting with antiserum which recognizes MuLV CA. Initial experiments were performed on the FeLV-infected cell line FL-74 (34) which showed that P+:ISP but not P+:PSI exerts a modest inhibitory effect on virus release (data not shown). Figure 6 shows the effects of three synthetic peptides and murine alpha-beta interferon on virus release from Rauscher MuLV-infected cells. Figure 6A shows the results of a reverse transcriptase assay which indicates that P+:ISP but not P+:PSI or ISP:P+ inhibited virus release at peptide concentrations of 25 ,ug/ml or higher. Murine interferon also inhibited virus release but at much lower concentrations (25 U/ml or 6.25 ng/ml). Figure 6B shows the entire length of two lanes from representative

BIOLOGICAL ACTIVITIES OF TM-DERIVED SYNTHETIC PEPTIDE

VOL. 64, 1990

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Peptide Conc (g/ml) FIG. 3. P+:ISP alters [3H]thymidine uptake by mitogen-activated splenic T cells. Percent positive control was defined as 100 x (experimental counts per minute)/(cells with mitogen but no peptide). The SI of each stimulated cell population without peptide is shown in parenthesis. (A) Splenocytes enriched by monoclonal antibody-mediated cytolysis. Symbols: 0, unfractionated cells (SI = 82.0 + 5.5);*, Lyt2.2-depleted cells (SI = 64.0 3.4); O, L3T4-depleted cells (SI = 26.1 + 5.3); 0, Lytl.2-depleted cells (SI = 14.9 + 3.4). (B) Splenocytes sequentially fractionated by nylon wool column (NWC) depletion and monoclonal antibody-mediated cytolysis. Symbols: 0, unfractionated cells (SI = 71.0 5.4); *, NWC-depleted cells (SI = 79.6 7.2); *, NWC- and Lyt2.2-depleted cells (SI = 336.9 + 43.7); 0, NWC- and Lytl.2-depleted cells (SI = 170.7 24.4). ±

±

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immunoblots in which the antiserum was tested for antigen specificity by the addition or omission of competing synthetic peptide. Figure 6C shows portions of four different immunoblots in which the cells had been exposed to P+:ISP (blot a), P+:PSI (blot b), ISP:P+ (blot c), or alpha-beta interferon (blot d). The results of the immunoblots confirm that P+:ISP and alpha-beta interferon inhibit virus release from this cell line. Results of a similar experiment are shown in Fig. 7 with Moloney MuLV-infected cells. Figure 7A shows results of a reverse transcriptase assay which indicates that P+:ISP but not P+ :PSI or alpha-beta interferon inhibited virus release at 125 100

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Peptide Conc (g/ml) FIG. 4. P+:ISP inhibits hemolytic plaque formation by SRBCprimed splenocytes. Percent positive control was defined as 100 x (number of experimental plaques)/(number of plaques from cells with SRBC but no peptide). Symbols: 0, P+:ISP; 0, P+:PSI.

peptide concentrations over 25 ,ug/ml. Figure 7B shows the entire length of two lanes from representative immunoblots in which the antiserum was tested for antigen specificity by the addition or omission of competing synthetic peptide. Figure 7C shows portions of three different immunoblots in which the cells had been exposed to P+:ISP (blot a), P+:PSI (blot b), or alpha-beta interferon (blot c). The results of the immunoblots confirm that P+:ISP but not P+:PSI or alphabeta interferon inhibited virus release from this cell line. DISCUSSION Sequence alignments of envelope precursor proteins from diverse type C and several type D retroviruses reveal a highly conserved sequence within the region of the TM (3). Comparison of retrovirus envelope sequences shows that Gln-Asn-Arg-Arg-Gly-Leu-Asp-Xnn-Leu is a conserved sequence in 20 envelope proteins which include the human T-lymphotropic virus types I and II and the bovine leukemia virus, FeLV, and MuLV. A 17-amino-acid peptide synthesized across the highly conserved sequence of MuLV TM, called by the authors CKS-17, displays biological activities reported for intact TM only if the peptide is chemically cross-linked to bovine serum albumin (2). The minimum peptide sequence essential for biological activity has been recently deduced to be an eight-residue sequence (29) very similar to the consensus peptide. We found that synthetic attachment of a CKS-17 homolog from FeLV TM to another short TM-derived sequence particularly rich in polar positive residues yielded a 29-amino-acid peptide, called in this report P+:ISP, which displayed some but not all of the biological activities reported for intact TM and albuminlinked peptide. Diverse in vitro activities of albumin-linked CKS-17 include inhibition of [3H]thymidine uptake by IL-1- and IL2-responsive cell lines (2, 9, 17, 29), inhibition of [3H]

J. VIROL.

WEGEMER ET AL.

1434

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FIG. 5. Similarities in protein sequence between the ISP domain of FeLV TM and a region of human (hu) alpha7 (32), murine (mu) alpha1 (1), and human alpha2 (21) interferons essential for antiviral-cytostatic activity.: indicates an identical amino acid match. indicates one of the following equivalents: Ala = Ser = Thr; Asp = Glu; Asn = Gln; Arg = Lys; Iso = Leu = Met = Val; Phe = Tyr = Trp. .

thymidine uptake by one- and two-way mixed leukocyte reactions, inhibition of antibody production as measured in a hemolytic plaque assay (25), inhibition of monocyte responsiveness to chemotactic peptides (11), and stimulation of natural killer cell activity (12). We found that the P+:ISP partially blocks [3H]thymidine uptake by concanavalin Astimulated, unfractionated splenocytes to a maximal extent of 30 to 50% at final peptide concentrations between 12.5 ,ug (3 ,uM) and 75 .ig (19 FLM) per ml of growth medium. At higher peptide concentrations, stimulation of [3H]thymidine uptake occurred. Monoclonal antibody-mediated cytolysis followed by concanavalin A stimulation indicated that T helper-inducer but not cytotoxic T cells are essential cellular components for the peptide-mediated effects. The P+:ISP slightly inhibited at high peptide concentrations but did not stimulate [3H]thymidine uptake by concanavalin A-activated

thymocytes. Antibody production by immunized splenocytes as measured in the T-helper-cell-dependent hemolytic plaque assay was also suppressed. Based on the apparent specificity of P+:ISP for splenic T cells, it is likely that the peptide directly affects T cells or possibly T-cell interaction with other cellular or extracellular components. Unlike reports on the albumin-linked peptide, we detected no inhibition of [3H]thymidine uptake by IL-13-stimulated U373 cells, one-way or two-way mixed leukocyte reactions (human or murine), and only a slight cytotoxic effect on IL-2-stimulated CTLL-2 cells. It is possible that P+:ISP expresses only a limited subset of biological activities reported for the albumin-linked peptide. Chemical crosslinking of peptide to albumin has been suggested to stabilize the IL-1-neutralizing activity of the peptide (2). The TM polar positive moiety of P+ :ISP may also stabilize biological

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Peptide Conc (.g/ml) Interferon Conc (U/ml) FIG. 6. Synthetic peptide and alpha-beta interferon effects on cellular release of virus from Rauscher MuLV-infected fibroblasts. (A) Reverse transcriptase assays of released virus after cell exposure to 0, 25, 50, or 100 ,ug of peptide or units of interferon per ml of growth medium. Symbols: 0, P+:ISP; 0, P+:PSI; A, ISP:P+; *, alpha-beta interferon. (B) Full height of a representative immunoblot labeled with antiserum blocked (+) or unblocked (-) by synthetic peptide antigen. (C) Four immunoblots of cell extracts and released virus after exposure to 0 (lanes 1 and 5), 25 (lanes 2 and 6), 50 (lanes 3 and 7), or 100 (lanes 4 and 8) ,ug of peptide or units of interferon per ml. Lanes 1 to 4 are whole-cell extracts; lanes 5 to 8 are viral pellets derived from the corresponding cell samples 1 to 4, respectively. All blots were sequentially exposed to synthetic CA peptide antiserum and 125I-protein A. Blots: a, P+:ISP; b, P+:PSI; c, ISP:P+; d, alpha-beta interferon.

BIOLOGICAL ACTIVITIES OF TM-DERIVED SYNTHETIC PEPTIDE

VOL. 64, 1990

10

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100

Peptide Conc (pg/ml) Interferon Conc (U/ml) FIG. 7. Synthetic peptide and alpha-beta interferon effects on cellular release of virus from Moloney MuLV-infected fibroblasts. Similar experiment setup to that described in the legend to Fig. 6. (A) Reverse transcriptase assays of released virus after cell exposure to 0, 25, 50, or 100 ,ug of peptide or units of interferon per ml of growth medium. Symbols: 0, P+:ISP; 0, P :PSI; Fl, alpha-beta interferon. (B) Full height of a representative immunoblot labeled with antiserum blocked (+) or unblocked (-) by synthetic peptide antigen. (C) Three immunoblots of cell extracts and released virus after exposure to 0 (lanes 1 and 5), 25 (lanes 2 and 6), 50 (lanes 3 and 7), or 100 (lanes 4 and 8) ji.g of peptide or units of interferon per ml. Lanes 1 to 4 are whole-cell extracts; lanes 5 to 8 are viral pellets derived from the corresponding cell samples 1 to 4, respectively. All blots were sequentially exposed to synthetic CA peptide antiserum and 125I-protein A. Blots: a, P+:ISP; b, P':PSI; c, alpha-beta interferon.

activity of the immunosuppressive peptide but perhaps in a manner which confers different biological effects. The pathway used by retroviral TM and albumin-linked CKS-17 to indirectly block mitogenic signals of IL-1 and IL-2 is presently unknown (9). Comparison of the ISP domain from FeLV TM with all available cytokine sequences in the Protein Databank revealed similarities between the TM consensus sequence and a region of alpha interferons shown by others (1, 21, 32) to be essential for the antiviral and cytostatic activities. We report that P+:ISP specifically inhibited virus release from two MuLV-infected cell lines. The antiviral effects do not correlate with alphabeta interferon sensitivity and, for this reason, might indicate that the peptide blocks virus release in a manner unrelated to the antiviral mechanism of alpha interferon. Alternatively, these results might actually be consistent with P :ISP mimicking one but not both protein domains of alpha interferon which influence biological activity (1). Our results may also indicate that polar positive domains in alpha interferons influence cytokine biological activity. The exact mechanisms of action for P+:ISP, native retroviral TM, and alpha interferon are still unclear. It is possible that P+:ISP affects splenic T helper-inducer cells and virus release from MuLV-infected fibroblasts through entirely distinct pathways. Alternatively, the peptide might utilize a mechanism common to diverse cell types. The effects of P+:ISP on (2'-5')oligo(A) synthetase activity and other specific markers of interferon activity have not yet been examined. The signal pathway might involve peptide interaction with functionally related or widely distributed receptors such as integrins. The interaction of leukocyte adhesion protein LFA-1 with ligand is intimately associated with T-cell activation (4). Another report showing that monoclonal antibodies to leukocyte adhesion protein LFA-1 block human immunodeficiency virus infection strongly implicates integrins as a receptor for retrovirus (14). The synthetic

peptide-cell interaction might also involve a common protein associated with several different receptors. Examples of receptor-associated proteins include the major histocompatibility complex class I molecules whose interaction with insulin and luteinizing hormone receptors precede intracellular signaling (10, 33) and epidermal growth factor receptor association with coated pit proteins (23). The identification of functionally important protein domains and the availability of biologically active synthetic peptides should prove useful in defining potentially shared mechanisms of action between retroviral TM and the antiviral-cytostatic-inducing domains of alpha interferons. ACKNOWLEDGMENT We thank Robert Naso for valuable discussions and technical advice. LITERATURE CITED 1. Bosveld, J., K. van As, A. C. P. Hekman, M. van Heuvel, J. Trapman, and E. C. Zwarthoff. 1989. The biological activity of interferon alpha is influenced by two distinct regions in the protein. Biochem. Biophys. Res. Commun. 164:22-29. 2. Cianciolo, G. J., T. D. Copeland, S. Oroszlan, and R. Snyderman. 1985. Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins. Science 230:453-455. 3. Cianciolo, G. J., R. J. Kipnis, and R. Snyderman. 1984. Similarity between plSE of murine and feline leukaemia viruses and p21 of HTLV. Nature (London) 311:515. 4. Dustin, M. L., and T. A. Springer. 1989. T-cell receptor crosslinking transiently stimulates adhesiveness through LFA-1. Nature (London) 341:619-624. 5. Elder, J. H., and J. I. Mullins. 1983. Nucleotide sequence of the envelope gene of Gardner-Amstein feline leukemia virus B reveals unique sequence homologies with a murine mink cell focus-forming virus. J. Virol. 46:871-880. 6. Emini, E. A., J. V. Hughes, D. S. Perlow, and J. Boger. 1985.

1436

7.

8.

9.

10.

11.

12.

13. 14.

15. 16. 17.

18.

19. 20.

21.

WEGEMER ET AL.

Induction of hepatitis A virus-neutralizing antibody by a virusspecific synthetic peptide. J. Virol. 55:836-839. Fan, H., and M. Paskind. 1974. Measurement of the sequence complexity of cloned Moloney murine leukemia virus 60 to 70S RNA: evidence for a haploid genome. J. Virol. 14:421-429. Garnier, J., D. J. Osguthorpe, and B. Robson. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120:97-120. Gottlieb, R. A., W. J. Lennarz, R. D. Knowles, G. J. Cianciolo, C. A. Dinarello, L. B. Lachman, and E. S. Kleinerman. 1989. Synthetic peptide corresponding to a conserved domain of the retroviral plSE blocks IL-1-mediated signal transduction. J. Immunol. 142:4321-4328. Hansen, T., J. Stagsted, L. Pedersen, R. A. Roth, A. Goldstein, and L. Olsson. 1989. Inhibition of insulin receptor phosphorylation by peptides derived from major histocompatibility complex class I antigens. Proc. Natl. Acad. Sci. USA 86:3123-3126. Harrell, R. A., G. J. Cianciolo, T. D. Copeland, S. Oroszlan, and R. Snyderman. 1986. Suppression of the respiratory burst of human monocytes by a synthetic peptide homologous to envelope proteins of human and animal retroviruses. J. Immunol. 136:3517-3520. Harris, D. T., G. J. Cianciolo, R. Snyderman, S. Argov, and H. S. Koren. 1987. Inhibition of human natural killer cell activity by a synthetic peptide homologous to a conserved region in the retroviral protein, p1SE. J. Immunol. 138:889-894. Hause, W. R., and R. G. Olsen. 1981. Clinical aspects of feline leukemia diseases, p. 89-114. In R. G. Olsen (ed.), Feline leukemia. CRC Press, Inc., Boca Raton, Fla. Hildreth, J. E. K., and R. J. Orentas. 1989. Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation. Science 244:1075-1078. Hopp, T. P., and K. R. Woods. 1981. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78:3824-3828. Jameson, B. A., and H. Wolf. 1988. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput. Appl. Biosci. 4:181-186. Kleinerman, E. S., L. B. Lachman, R. D. Knowles, R. Snyderman, and G. J. Cianciolo. 1987. A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocytemediated killing by inactivating interleukin 1. J. Immunol. 139:2329-2337. Kloetzer, W., R. Kurzrock, L. Smith, M. Talpaz, M. Spiller, J. Gutterman, and R. Arlinghaus. 1985. The human cellular ABL gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity. Virology 140:230-238. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Lafrado, L. J., M. G. Lewis, L. E. Mathes, and R. G. Olsen. 1987. Suppression of in vitro neutrophil function by feline leukaemia virus (FeLV) and purified FeLV-pl5E. J. Gen. Virol. 68:507-513. Lydon, N. B., C. Favre, S. Bove, 0. Neyret, S. Benureau, A. M. Levine, G. F. Seelig, T. L. Nagabhushan, and P. P. Trotta. 1985. Immunochemical mapping of a-2 interferon. Biochemistry 24: 4131-4141.

J. VIROL. 22. Mathes, L. E., R. G. Olsen, L. C. Hebebrand, E. A. Hoover, and J. P. Schaller. 1978. Abrogation of lymphocyte blastogenesis by a feline leukaemia virus protein. Nature (London) 274:687-689. 23. Mayo, K. H., M. Nunez, C. Burke, C. Starbuck, D. Lauffenburger, and C. R. Savage, Jr. 1989. Epidermal growth factor receptor binding is not a simple one-step process. J. Biol. Chem. 264:17838-17844. 24. Meyers, E. W., and W. Miller. 1988. Optimal alignments in linear space. Comput. Appl. Biosci. 4:11-17. 25. Mitani, M., G. J. Cianciolo, R. Snyderman, M. Yasuda, R. A. Good, and N. K. Day. 1987. Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses. Proc. Natl. Acad. Sci. USA 84:237-240. 26. Naso, R. B., Y. C. Wu, and C. A. Edbauer. 1982. Antiretroviral effect of interferon: proposed mechanism. J. Interferon Res. 2:75-96. 27. Olsen, R. G., L. E. Mathes, and S. W. Nichols. FeLV-related immunosuppression, p. 89-114. In R. G. Olsen (ed.), Feline leukemia. CRC Press, Inc., Boca Raton, Fla. 28. Orosz, C. G., N. E. Zinn, R. G. Olsen, and L. E. Mathes. 1985. Retrovirus-mediated immunosuppression. I. FeLV-UV and specific FeLV proteins alter T lymphocyte behavior by inducing hyporesponsiveness to lymphokines. J. Immunol. 134:33963403. 29. Ruegg, C. L., C. R. Monell, and M. Strand. 1989. Identification, using synthetic peptides, of the minimum amino acid sequence from the retroviral transmembrane protein pl5E required for inhibition of lymphoproliferation and its similarity to gp2l of human T-lymphotropic virus types I and II. J. Virol. 63: 3250-3256. 30. Ruggeri, Z. M., R. A. Houghten, S. R. Russell, and T. S. Zimmerman. 1987. Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets. Proc. Natl. Acad. Sci. USA 83:5708-5712. 31. Schmidt, D. M., N. K. Sidhu, G. J. Cianciolo, and R. Snyderman. 1987. Recombinant hydrophilic region of murine retroviral protein plSE inhibits stimulated T-lymphocyte proliferation. Proc. Natl. Acad. Sci. USA 84:7290-7294. 32. Shafferman, A., B. Velan, S. Cohen, M. Leitner, and H. Grosfeld. 1987. Specific residues within an amino-terminal domain of 35 residues of interferon a are responsible for recognition of the human interferon a cell receptor and for triggering biological effects. J. Biol. Chem. 262:6227-6237. 33. Solano, A. R., G. Cremaschi, M. L. Sanchez, E. Borda, L. Sterin-Borda, and E. J. Podesta. 1988. Molecular and biological interaction between major histocompatibility complex class I antigens and luteinizing hormone receptors or P-adrenergic receptors triggers cellular response in mice. Proc. Natl. Acad. Sci. USA 85:5087-5091. 34. Theilen, G. H., T. G. Kawakami, J. D. Rush, and R. J. Munn. 1969. Replication of cat leukaemia virus in cell suspension cultures. Nature (London) 222:589-590. 35. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 36. Wellman, M. L., G. J. Kociba, M. G. Lewis, L. E. Mathes, and R. G. Olsen. 1984. Inhibition of erythroid colony-forming cells by a Mr 15,000 protein of feline leukemia virus. Cancer Res. 44:1527-1529.