Topography of murine leukemia virus envelope proteins: characterization of transmembrane components.

0022-538X/83/061056-05$02.00/0

Copyright©1983,AmericanSocietyforMicrobiology

Topography

of Murine Leukemia Virus

Envelope

Proteins:

Characterization of Transmembrane Components

ABRAHAMPINTER* AND W. J.HONNEN

MemorialSloan-Kettering Cancer Center, New York, New York 10021 Received 18 November1982/Accepted 21 March 1983

Trypsinization of intact Moloneymurine leukemiavirusresulted incleavage of p15(E) and Prl5(E)at asitenearthemiddle ofthemolecule, producing a 9,000-dalton amino-terminal fragmentwhich contains thedisulfide linkage site togp70 andwhichcarries p15(E) epitopes band c, but not epitope a. After solubilization of the viral membrane, trypsinization occurred at a second site within 1,000 daltons of the carboxy end ofp15(E). This site is not exposed in intact virions, indicating that p15(E)and Prl5(E)are transmembrane proteins.

Murine leukemia viruses (MuLVs) contain two major envelope proteins, gp70 and p15(E). These proteins are produced by cleavage ofa commonprecursor(2,8, 12, 16) and form disul-fide-linked complexes with each other (9, 11). Many observations indicate that gp70 is only loosely attachedtotheviral membrane,whereas p15(E) appears to be an integral membrane protein. Intracellularly, Prl5(E), a precursor form ofp15(E), is found which is usually proc-essed to p15(E) at a late step in the budding process (3); however, low levels of intact Prl5(E) are often incorporated into virions. Studies with monoclonal antibodies have indi-cated thatp15(E)containsatleast threedistinct antigenic epitopes, a,b,andc.Sitea isspecific forecotropicvirus,site bispresentinecotropic andxenotropicMuLVs, and sitecis conserved as well for feline leukemia virus (6). Recently, theamino acidsequences of theseproteins have been deduced from the DNA sequence of the envgene(13). This sequenceindicates that Mo-loneyPrl5(E) is 196 amino acidslong,whereas maturep15(E)contains 180 residues. Hydropho-bicregionsarepresent near each end ofp15(E): a42-residuestretchoccupies positions2through 43, and a 30-residue stretch occupies positions 135through 164. Both of theseregions arelong enoughtospanthemembrane, and thusseveral possible models for the association of these proteins withthemembranecanbe constructed. Inthispaper, we presentevidence of the orien-tations of

p15(E)

Prl5(E)

Treatment of freshly purified intact virions with high concentrations of trypsin resulted in thecomplete degradationofgp7O andin alarge diminution in the levels of

p15(E)

(Fig.1).Fragments of gp7O andan approximate-ly 9,000-dalton (9K) fragment of p15(E), recog-nized specifically by monoclonal antibody 42/114 (lanes C) butnotbyantibody9-E8(lanes B), were formed. (9-E8 recognizes epitope a; 42/114has areactivitypattern similartothatof 19-F8 in an analysis against a broad panel of virus isolates, and thus we tentatively assigned thespecificityof thisantibodytoepitope c.)The concentrations of the internal proteins p30 and p12remainedunchanged,confirmingthe integri-tyof the viralenvelope.These results confirmed a previous report that the env proteins gp7O, Prl5(E), andp15(E) are all exposed atthe

sur-face of intact virions (7) and further suggested that theaccessibilityofgp7Ototrypsinisgreater than thatofp15(E).

The relative sensitivityofp15(E) to trypsini-zation in intact versus solubilized virus was compared(Fig. 2A). The titration indicated that the susceptibility ofgp7O tocleavage isgreater after lysis, whereas the extent and kinetics of cleavageofp15(E)arecomparablefor intact and lysedvirions. From otherstudies,weknow that the major gp7O fragments obtained are derived fromthecarboxy terminus ofthemolecule,and there appears to be a relationshipbetween the reduction in size of the gp7Ofragments formed andthecleavage ofp15(E), suggestingthat deg-radation of the carboxyl domain of gp7O may increase theaccessibility ofp15(E) to proteoly-sis. After treatment with the highest trypsin concentration, a slight shift in the mobility of p15(E) was detected forthe solubilized protein but not for the intact virus (lanes 3). This size shift is more clearly visualized in Fig. 2B and correspondsto amolecularweight changeof500 to 1,000. Analysis ofthe predicted amino acid sequence ofMoloney p15(E) indicated that the

1056

on November 10, 2019 by guest

http://jvi.asm.org/

NOTES 1057

CONTRC'OL +TRYPSIN

; A 3 CC E v A B C D E

,J7-)- _

Pr

15(E)-p15(E;

pl2 Om 'A D^

Uo

FIG. 1. Effect of trypsinization of intact Moloney MuLV. Freshly purified [35S]methionine-labeled viri-ons (200,000 cpm per sample) were incubated with trypsin(1.25 mg/ml) for30at 37°C. The trypsinwas inactivated with Trasylol(1,000U/ml;Bayer),and the virionsweresolubilizedwith 0.5% Nonidet P-40. Both treated and nontrypsinized (control) samples were thensolubilized withradioimmunoprecipitation buffer (0.01MTris buffer[pH 7.4] 0.5% NonidetP-40,0.5M NaCl) andimmunoprecipitated with the following anti-sera: lanes A, hyperimmune goat otRauscher gp70 serum; B, monoclonal otpl5(E) antibody 9-E8 (6); C, monoclonal apl5(E) antibody 42/114; D, rabbit otR serum, which specifically recognizes Pr15(E) butnot p15(E) (15); and E, normalgoatserum. Immunopreci-pitates were solubilized by being boiled with 1% sodiumdodecyl sulfate-1% mercaptoethanol and an-alyzedon a12%polyacrylamide gel by the method of Laemmli (4). Lanes v represent the complete virus samples.Analysisof theseimmunoprecipitates under nonreducing conditions indicated that the coprecipita-tionofp15(E) by the agp70serum(lanesA)wasdue in parttothepresenceofdisulfide-linked complexesand in part to the noncovalent association of these pro-teins.

conversion to this 14K fragment must involve cleavage at the carboxy terminus of p15(E),

astherearebasic aminoacid residues 8 and 10

residues from the carboxy end (assumed to be

residue 180[13]), whereas thefirsttrypsinsiteat

theamino end of the moleculeoccursatposition 48 (see Fig. 5A). The accessibility of this site

only after solubilization of the viral particles stronglysuggests that this region ofthe protein is located inside the viral membrane. This

car-boxy-terminal region immediately follows the

hydrophobic sequence at positions 135 to 164, thus implicating that region as the

transmem-brane segmentof the protein.

Thistrypsin site nearthecarboxy terminus of p15(E) provided us with a specific marker for

that end of the protein. The fact that an identi-cally sized 9Kfragmentwasformed upon tryp-sinization of both intact and solubilized virions (Fig. 2B)indicated that this fragment could not

be derived from the carboxy end of the mole-cule. Consideration of the fragment size and of the predicted distribution of trypsin sites on p15(E) (see Fig. 5A),ledus toconcludethat the 9K band corresponds to the amino-terminal do-main ofthe molecule.

We have previously shown that p15(E) is disulfide bonded to a site in the carboxy-termi-nal domain ofgp7O (10). To determine whether thecysteine(s) ofp15(E)involved inthislinkage is located in the 9K fragment, we examined the association of this band with gp7O fragments from virus samples in which disulfide bonds betweengp7Oandp15(E) had been stabilized by treatment with N-ethylmaleimide (NEM) (11). The 9K band was efficiently coprecipitated by agp70 serum from NEM-treated virus, but not from virus for which the NEM treatment was omitted, indicatingthat it is disulfide linked to gp7O (Fig. 3). Consistent with this conclusion, the bulk of the coprecipitated 9K band was not seen upon analysis of this sample under nonre-ducing conditions (data not shown). It thus appears thatcoprecipitationof the 9K fragment by agp70 sera is dependent upon disulfide bond formation withgp7O, and unlike the situation for intact p15(E) (Fig. 1A), noncovalent associa-tions do notplay amajorrole. Further confirma-tion of the disulfide linkage of the 9K band to gp7O was provided byanalysisof the reactivities of four monoclonal aplS(E) antibodies towards 125I-labeled pl5(E)-gp7O disulfide-linked frag-mentsfrom AKR MuLV(Fig.4). Trypsinization generated a 32K carboxy-terminal fragment of gp7O, which remained disulfide linkedtop15(E) and to the p15(E) 9K fragment also formed, resultinginresolution undernonreducing condi-tionsof a 47K bandanda41Kband, present in roughly equivalent amounts. Inagreementwith ourprevious results, both the 47Kcomplexand the 41K complex were recognized by 42/114, whereas 9-E8 recognized only the 47K band. Antibodies 19VIII-E8 and 19-F8, directed against p15(E) epitopes b and c(6), also recog-nized the 41K band, indicating that these epi-topes are alsolocated in the 9Kamino-terminal p15(E) fragment. Theseresultsare in agreement with the observations by Stone and Nowinski (14) that antibodies against epitopes b and c exhibitbinding competitionwitheachother but not withantibodies against epitopea.

In addition to the 9K band recognized by 42/114, aslightly larger fragment wasfound in variable amounts in immunoprecipitates ob-tained with antibodies 42/114 and 9-E8 (Fig. 2 and 3). This band, corresponding to afragment VOL 46, 1983

on November 10, 2019 by guest

http://jvi.asm.org/

1058 NOTES

A

N

h ,r LY' SE [I

i;" c.l'i[gpt.. .p15(L

2 3 2 3

gp7O1-- -

_S-..~~.4

Prl5lE)- C

p15(E)-pl5(E) p12-_

0

04 e4 a

B

2 3 4

v I L I L IL

II'

4040s fwt

I.0.a .*&i

i

e

e

-FIG. 2. Comparison of trypsin sensitivity ofgp70and p15(E) inintact and lysedvirions. Freshly purified [35S]methionine-labeled Moloney MuLV, either intact oraftersolubilization, was treated withtrypsin. After neutralization of trypsin with Trasylol, the samples were diluted with radioimmunoprecipitation buffer, immunoprecipitatedwith9-E8,andanalyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. (A) Thefollowing trypsin concentrationswereused:lanes 1, 62.5 p.g/ml; 2,250,ug/ml;and3,1mg/ml. (B) A trypsin concentrationof 1 mg/mlwasused,andsampleswereimmunoprecipitatedwiththefollowing antisera: lanes 1, hyperimmune goataRauscher gp70; 2, monoclonal apl5(E) antibody 9-E8; 3, monoclonalapl5(E) antibody 42/114;and4,normalgoatserum.

+NEM

Uncleoved +Trypsin_

2 3 4 1 2 3 4

_p x X...

- NEM +Trypsin

2 3 4

ofapproximately 10K, was identical in size for

both intact and solubilized virions and was

formed ingreateryield for the solubilized virions (Fig. 2B). That the size of this band was not

decreased after solubilization indicates that it

cannotbe a carboxy-terminal p15(E) fragment.

FieHa ,. Non'reruced

34 -1 4 5

___ _

W.--_ _ __m 4m -14K

a*s-a -9 K

FIG. 3. Coprecipitationofthep15(E)9Kfragment by axgp70 serum. [35S]methionine-labeled Moloney MuLVwastreated with NEM(0.1%,wt/vol)for 15h, solubilized with0.5% Nonidet NP-40, and treated with trypsin (1.25 mg/ml). Control samples for which the trypsin or NEM treatments were omitted were also analyzed. After immunoprecipitation, samples were analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresison12%gels. Antisera used: lanes 1, goat oaRauscher gp7O; 2, 9-E8; 3, 42/114; and 4, normalgoatserum.

-47 K -41 K

32K

FIG. 4. Coprecipitation of gp7O fragments with apl5(E) monoclonal antibodies. 125I-labeled AKR MuLV(106 cpm)wassolubilized, treatedwithtrypsin (250 ±tg/ml), and then immunoprecipitated and ana-lyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10%o gels both before and after reduction with mercaptoethanol. Antibodies used: lanes1, 42/114; 2, 9-E8;3, 19-F8; and 4,19VIII-E8.

14 K

- 9K

-SW I-W

0

on November 10, 2019 by guest

http://jvi.asm.org/

Itmightrepresent a largeramino-terminal frag-ment,whichmaybea precursor tothe 9Kband. Ifso, this would indicate thatepitope a recog-nized by antibody 9-E8 is located near the carboxy terminus of the10Kfragment.An alter-nate possibility is that the 10K fragment may represent a gp7O fragment which is strongly associated withp15(E) and isthus coprecipitat-ed by ap15(E) antibodies. Clarification of the structureandorigin of thisfragment will require analysis of these components by peptide map-ping.

The conclusions derived from this study are

summarized in diagrammatic form in Fig. SB. Bothp15(E)andPrl5(E)areintegralmembrane proteins, with a carboxy-terminal hydrophobic

sequence traversing the membrane (a similar model has beenproposedby Lenzetal., based onthededuced amino acidsequenceof the Akv env gene [5]). The transmembrane region is drawn as a helix, because a consideration of energetics has indicated that this is the most

stable form for a polypeptide in the apolar portion of membrane bilayers(1). A linearmap, drawn to scale, of the distribution of charged residues on p15(E) indicates the presence of many potential trypsin sites in the central por-tion of thep15(E) protein which could account for the formation of the observed fragments (Fig. 5A). At least one trypsin-sensitive site is exposed on the surface of intact virions, al-thoughcleavageatthis siterequiredhigh trypsin

A

* - .0 4

* *

I I I I I

100 Io

t

p15(E) PrI5CE)

B

pp70

HOOC *

FIG. 5. Diagrammatic representation of structural features and membrane orientation of Prl5(E).(A)Linear mapindicating location of charged residues of Prl5(E)(3). Every 10thresidue is marked off.Positively charged

residues(lysines and arginines)arerepresentedbyadot above the line;negativelychargedresidues (aspartic and

glutamic acids)arerepresented byadot below the line.The carboxy termini of p15(E) and Prl5(E)areindicated

byarrows.(B) Diagram illustrating membrane orientation ofPrl5(E).Singlearrowheadsrepresenttrypsin sites

involved in formation of 9K and 14K fragments; the double arrowhead represents the natural cleavage site

utilized inprocessing Prl5(E)top15(E).The carboxy-terminalnonpolar region of p15(E) is presented traversing

themembrane inhelical conformation. The heavy linesrepresentthenonpolar regionsattheamino terminus of

p15(E) and carboxy terminus of gp7O, which may participate in the noncovalent association of these two molecules.

RESIDUE NO:

I a I I

so

0 * 0 de 0 0 0 0 00 09 0 00 0 0 0 * so 0 0 0 4

on November 10, 2019 by guest

http://jvi.asm.org/

concentrations, suggesting that accessibility to the protease is somewhat blocked, presumably owing to the proximity ofgp7O. The deduced sequenceofp15(E) indicates that therearethree cysteines in the amino-terminal half of Pr15(E) atpositions 86, 93, and 94 (13), oneor more of whichmustbeinvolved in this disulfide linkage. Anadditional trypsin site is located in the cyto-plasmic region ofp15(E); this site is accessible only after solubilization of the viral membrane. TheDNAsequenceofthe env geneindicates the presence of another hydrophobic region at the amino terminus of p15(E), and one near the carboxy terminus ofgp7O (13).Amodel inwhich these regions alsotraversethe bilayeris incon-sistent with the knownperipheralassociationof gp7O with the membrane. These regions maybe involved in noncovalent associations between thetwoproteins.

Thecytoplasmic location of the carboxy-ter-minal domain ofPrl5(E) suggeststhepossibility that these sequences may be recognized by the viralgagproteins andthereby provide the asso-ciation betweengagandenvproteinsnecessary for assemblyof infectious virions. If this is the case, then the reported hypervariability at the extremecarboxyterminusofPrl5(E) startingat residue191 (5),would indicate that thisregion is notcritical for such interactionsbut, rather,that the conserved residues 165-190of the cytoplas-mic domainmaybe involved.

This researchwassupportedbyPublic HealthService grant CA-16599 from the National Institutes of Health.

LITERATURE CITED

1. Engelman, D. M., and T. A.Steitz.1981. Thespontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23:411-422.

2. Famulari, N. G., D. L.Buchhagen, H.-D.Klenk,and E. Fleissner. 1976. Presenceof murine leukemia virus enve-lope proteinsgp7Oandp15(E)in a commonpolyprotein of infected cells. J. Virol. 20:501-508.

3. Green, N., T.M.Shinnick, 0. Witte,A.Ponticelli, J. G.

Sutcliffe, and R. A. Lerner. 1981.Sequence-specific anti-bodies show thatmaturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-termi-nalpeptide. Proc. Natl. Acad. Sci. U.S.A. 78:6023-6027. 4. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London) 227:680-685.

5. Lenz, J., R. Crowther, A. Straceski, and W. Haseltine. 1982. Nucleotidesequence of the Akv env gene. J. Virol. 42:519-529.

6. Lostrom,M.E.,M. R.Stone, M. Tam, W.N.Burnette,A.

Pinter, and R. C. Nowinski.1979.Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p15(E) and gp7O envelope proteins. Virology 98:336-350.

7.Montelaro, R. C., S. J. Sullivan, and D. P. Bolognesi. 1978. An analysis of type-C retrovirus polypeptides and their associations in the virion. Virology 84:19-31.

8. Naso, R. B., L. J Arcement, W. L. Karshin, G. A. Janjoom, and R. B. Arlinghaus. 1976. A fucose-deficient glycoprotein precursor to Rauscher leukemia virus gp69/ 71. Proc.Natl. Acad.Sci. U.S.A. 73:2326-2330. 9. Pinter, A., and E. Fleissner. 1977. The presence of

disul-fide-linked gp70-p15(E) complexes in AKR MuLV. Virol-ogy 83:417-422.

10. Pinter, A., W.J.Honnen, J. S. Tung, P. V. O'Donnell, and U.Hammerling. 1982. Structural domains ofendogenous

murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies. Virology 116:499-516.

11. Pinter, A., J. Lieman-Hurwitz, and E. Fleissner. 1978. The nature of the association between the murine leukemia virus envelopeproteins. Virology91:345-351.

12. Shapiro, S. Z., M. Strand, and J. T. August. 1976. High molecular weight precursor polypeptides to structural proteins of Rauscher murine leukemia virus. J. Mol. Biol. 107:459-477.

13. Shinnick, T. M., R. A. Lerner, and J. G. Sutcliffe. 1981. Nucleotide sequenceof Moloney murine leukemia virus. Nature(London) 293:543-548.

14. Stone, M. R., and R. C. Nowinski. 1980. Topological

mappingofmurine leukemia virus proteins by competi-tion-binding assays with monoclonal antibodies. Virology 100:370-381.

15. Sutcliffe, J. G., T. M. Shinnick, N. Green, F. T. Liu, H. L. Niman, and R. A. Lerner. 1980. Chemicalsynthesis of a

polypeptide predictedfromnucleotide sequence allows detectionofa newretroviral geneproduct. Nature (Lon-don) 287:801-805.

16. VanZaane, D.,M.J.A.Dekker-Michielson,andH. P.J.

Bloemers. 1976.Virus-specific precursorpolypeptides in cellsinfected with Rauscher leukemia virus: synthesis, identificationandprocessing.Virology75:113-129.

J.VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/