Proc. Nati. Acad. Sci. USAVol. 80, pp. 5965-5969, October 1983Genetics

Construction and characterization of Moloney murine-leukemiavirus mutants unable to synthesize glycosylated gag polyprotein

(murine leukemia virus /recombinant DNA /glycosylated gag protein)

HUNG FAN, HILARY CHUTE, EUPHEMIA CHAO, AND MIRIAM FEUERMANDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717

Communicated by Peter K. Vogt, June 27, 1983

ABSTRACT Murine leukemia virus (MuLV) encodes two in-dependent pathways for expression of the gag gene. One pathwayresults in processing and cleavage of the precursor Pr65m to yieldthe internal capsid proteins of the virion and is analogous to gagpolyprotein precursors for all classes of retroviruses. The otherpathway, which is not encoded by several other classes of retro-viruses, begins with a glycosylated polyprotein gPr8 a. gPr8agis synthesized independently of Pr65Pa9; it contains Pr65gag pep-tides and additional amino-terminal protein. It is modified by fur-ther addition of carbohydrate, exported to the cell surface, andreleased from the cell but does not appear in virus particles. Toinvestigate the role of glycosylated gag in MuLV infection, twomutants of Moloney MuLV (M-MuLV) deficient for synthesis ofgPr809a9 but able to synthesize Pr65"ag were constructed. Themutants were obtained by substitution into a molecular clone ofM-MuLV DNA by DNA from two acutely transforming viruses,Ableson MuLV (Ab-MuLV) and Moloney murine sarcoma virus(M-MSV). Both Ab-MuLV and M-MSV are derived from M-MuLVand they express M-MuLV gag sequences, but some strains do notsynthesize glycosylated gag protein. For Ab-MuLV, a 177-base-pairPst I fragment from the P90 strain containing the initiation codonfor Pr659ag was substituted for the equivalent fragment in M-MuLVDNA. For M-MSV, 1.5 kilobases at the 5' end of the genome wassubstituted. Transfection of the recombined DNAs onto NIH-3T3cells produced infectious M-MuLV, although the infected cells didnot produce gPr809a9. Therefore glycosylated gag is not absolutelyrequired for MuLV replication. Deletion of the glycosylated gagpathway did not significantly reduce the level of virus production,although a minor difference in XC plaque morphology was ob-served.

Retroviruses contain three genes for replication: gag, pol, andenv, which encode polyprotein precursors for the internal cap-sid proteins, reverse transcriptase, and envelope glycoprotein,respectively (1). Murine leukemia virus (MuLV) differs frommost other retroviruses in that it encodes two different path-ways for gag gene expression (2). These two pathways beginwith two independent translation products, gPr80gag and Pr65ag.Pr65gag is processed by proteolytic cleavage to yield the internalcapsid proteins of the virus particle and is analogous to the gagpolyprotein precursors of other retroviruses (3-5). gPr8Oggcontains the amino acid sequences of Pr65gag as well as 4-6 kilo-daltons (kDa) of amino-terminal protein (6, 7). The additionalamino-terminal peptides result in glycosylation of gPr8O'ag duringtranslation (8, 9). gPr8 gag is processed by the further additionof carbohydrate and exported to the cell surface where it ap-pears as a glycoprotein of 95 kDa (8-11). It may be also be re-leased into the extracellular medium as cleavage products of 55and 40 kDa (8, 12). Glycosylated gag products are not incor-

porated into MuLV virions, but they associate with the extra-cellular matrix (13).

While the function of Pr65gag is known, the role of gPr809aein MuLV infection is not known. Glycosylated gag might pro-vide some function required for viral replication, or it mightplay an accessory role. We previously isolated mutants of Mo-loneyMuLV (M-MuLV)-infected mouse fibroblasts that did notexpress gag proteins at the cell surface, and they were deficientfor virus production. However, these mutants were cellular,not viral, in nature and they produced normal amounts ofgPr8g'ag intracellularly (14). To obtain a more definitive an-swer, we constructed two mutants of M-MuLV at the recom-binant DNA level and recovered virus by transfection. The con-structions. and characterizations of the mutant viruses aredescribed here.

MATERIALS AND METHODSCells and Viruses. All cells were grown in Dulbecco-mod-

ified Eagle's medium/10% calf serum; Mouse NIH-3T3 cellswere described previously (15), as were M-MuLV-infected NIH-3T3 cells (clone A9) (16).

The UV-XC assay for MuLV was carried out as described (17).Assay of viral reverse transcriptase by the addition of exoge-nous poly(rA):oligo(dT) template-primer (16) and banding ofvirus in sucrose density gradients (18) was carried out as de-scribed.

Recombinant DNA Cloning. A phage recombinant DNAclones (A-MLV clones 48, 61, and 63) carrying integrated copiesof M-MuLV provirus were described previously (19). pMSV-1is a plasmid clone of unintegrated Moloney murine sarcomavirus (M-MSV) DNA permuted about the unique HindIII site(20). pSLT is a subclone of pMSV-1 deleted from the Sal I sitein M-MSV DNA to the Sal I site in the pBR322.vector (see Fig.2) and was kindly provided by Inder Verma. P90(Abl), a plas-mid clone of unintegrated Abelson MuLV (Ab-MuLV) (P90 strain)DNA, was kindly provided by Owen Witte. Plasmid vectorpBR328 has been described by Soberon et al. (21).

Restriction fragments were purified by electrophoretic elu-tion from agarose gels onto diethylaminoethyl (DEAE)-paper(22) or onto DEAE-cellulose membranes (NA45, Schleicher &Schuell) followed by recovery by high-salt elution. Ligation ofvector and insert fragments was carried out with T4 DNA ligaseunder standard conditions (23). In cloning steps in which theplasmid vector could recircularize without acquisition of a DNAinsert, the vector was treated with bacterial alkaline phospha-tase prior to ligation (23). Transformation of ligation reactions

Abbreviations: MuLV, murine leukemia virus; M-MuLV and Ab-MuLV,Moloney and Abelson strains of MuLV, respectively; kDa, kilodalton(s),M-MSV, Moloney murine sarcoma virus; bp, base pair(s); kb, kilo-base(s).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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into Escherichia coli C600 was carried out by the calcium shockprocedure (23). For some steps, screening of transformed bac-teria was by colony hybridization with nick-translated insert DNAand, for other steps, minipreparations of plasmids (24) from in-dividual colonies were analyzed for diagnostic restriction frag-ments.

Transfections. Transfection of DNA ligation reaction mix-tures or of plasmid DNA onto NIH-3T3 cells was essentiallyaccording to Copeland et al. (25) as modified by Lai and Verma(26). Briefly, NIH-3T3 cells were seeded at 106 cells per 5-cmtissue culture dish, and the following day a calcium phosphateprecipitate of DNA was added. After incubation for 8 hr, thecells were treated for 45 sec with 20% dimethyl sulfoxide, fur-ther incubated for 1 day in growth medium, and serially pas-saged at a 1:5 dilution every 3 days. One dish from each trans-fer was assayed for virus production by the UV-XC cell assayon reaching confluency.

Immunoprecipitations. Cell cultures were washed with warmTris-buffered saline, and 1 ml of Tris-buffered saline containing100 ACi of [.S]methionine (1 Ci = 37 GBq) was added to each9-cm dish. The cultures were incubated at 370C for 1 hr withoccasional rocking. At the end of the labeling period, cyto-plasmic extracts were prepared, immunoprecipitated with rab-bit anti-p30 serum, and analyzed by NaDodSO4/polyacryl-amide gel electrophoresis as described (8).

RESULTSConstruction of Recombinant DNA Clones Mutant in

gPr8Om. Two facts facilitated construction of recombinant DNAclones unable to synthesize gPr809aa but able to synthesizePr659ag. First, the entire nucleotide sequence of M-MuLV DNAhas been determined, and the precise location of the regioncoding for Pr65SaS is known (27). In particular, Pr655ag trans-lation starts at an initiation codon contained within a 177-base-pair (bp) Pst I fragment located between RNA nucleotides 567and 743 (Fig. 1A). Second, two acute transforming viruses, Ab-MuLV and M-MSV, arose by recombination with M-MuLV. Both

APs Ps X

LTR Pr659"g

of these viruses have been molecularly cloned (20, 28) and havemany restriction sites in common with M-MuLV DNA. Fur-thermore, some strains of these viruses are deficient in the abil-ity to encode glycosylated gag proteins.

Construction of a gPr80gag-negative mutant by recombina-tion with the Ab-MuLV genome is diagrammed in Fig. 1B. Thetransforming protein of Ab-MuLV is a polyprotein containingamino-terminal M-MuLV gag sequences fused to abl oncogenesequences (29). Several strains of Ab-MuLV encode gag-abl fu-sion proteins ranging in size from 150 to 90 kDa. Some strains(including the P150gag-abl strain) encode both glycosylated andnonglycosylated fusion proteins, presumably representing fu-sion of the amino termini of OM gag and Pr655gag to abl pro-tein. However, other strains (such as the P90 and P120 strains)encode only nonglycosylated gag-abl polyprotein, suggestingthat sequences encoding the amino termini of gPr8g5ag are al-tered in these strains. D. Robertson and 0. Witte (personalcommunication) have observed that the 177-bp Pst I fragmentof DNA from the P90. strain of Ab-MuLV (analogous to the 177-bp Pst I fragment of M-MuLV) contains a base change resultingin a termination codon in the open reading frame to the 5' sideof the Pr65gag initiation AUG. Therefore, the 177-bp fragmentof P90 Ab-MuLV was substituted for the 177-bp fragment ofM-MuLV at the recombinant DNA level (Fig. 1B). This wasachieved by a series of cloning steps involving subcloning of aHindIII fragment containing the 5' part of an integrated M-MuLV provirus into a plasmid vector lacking a Pst I site fol-lowed by deletion of the 177-bp Pst I fragment and reintroduc-tion of the Ab-MuLV 177-bp fragment. The resulting HindIIIfragment was then ligated in vitro with a HindHIl fragment con-taining the 3' part of an integrated M-MuLV provirus, and theligation mixture was directly transfected onto NIH-3T3 cells.The results are described below.

Construction of a gPr80gag-negative mutant by recombina-tion with the M-MSV genome is diagrammed in Fig. 2. Moststrains of M-MSV encode a nonglycosylated gag polyprotein of60-65 kDa, analogous to Pr65ga9 of M-MuLV, but none encode

HD

LTRM-MuLV provirus

A' XMLV Clone 63HD HD HD HDRI 7.5 kb , 4.5 D

P, -

Ps Ps

B~~~~~~HDHD

aMP tet~PstI+sl - A~%3Ps 'PyHindm

No

XMLV Clone 637.5 kb HindME

m Transfect NIH-3T3

FIG. 1. Substitution of Ab-MuLV sequences into M-RI MuLV DNA. (A) Restriction map ofM-MuLV DNA. The di--' -X rection of-transcription is indicated by the arrow. LTR, long

terminal repeat. The initiation codon for Pr65gw is withinthe 177-bpPst I fragment. (A') Restriction map forthe A-MLVclone 63 (19), a clone of a M-MuLV provirus integrated inmouse cell DNA. M-MuLV sequences within the insert areindicated by the hatched bar. AL, A Charon 4A left cloningarm; AR, right cloning arm. (B) Steps involved in substi-tution of the Ab-MuLV 177-bp Pst I fragment for the M-MuLVPst I fragment. (i) ThePst I site in pBR328 was elim-inated by digestion with Pst I, treatment with nuclease S1,and religation (pBR328 Pst-). This inactivated the ampi-cillin (amp)-resistance gene but not the chloramphenicol

figate (cam)- and tetracycline (tet)-resistance genes. The 7.5-kilo-base (kb)HindM fragment containing the 5' part ofM-MuLVproviral DNA from A-MLV clone 63 was then cloned intopBR328 Pst- at the HindIII site (p63-5'). The M-MuLV Pst

>8D I fragment was eliminated by cleavage with Pst I and re-ligation (p63-5'APst) followed by insertion ofthe Ab-MuLV177-bp Pst I fragment (p63-5'-Abl). Orientation of the Ab-MuLV Pst I fragment was determined by mapping a BstEIIsite located asymetrically within the Pst I fragment. The7.5-kb HindHI fragment from p63- 5'-Abl was ligated in vi-tro with the 4.5-kbHindIII fragment ofA-MLV clone 63 con-taining the 3' part of the M-MuLV provirus and directlytransfected into NIH-3T3 cells. HD, HindIl; Ps, Pst I; X,Xho I, RI, EccoRI.

XL

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- 4.7 kb - lX SmiK1Ii

XMLV Clone 48

X K KSmK KI -offIIIRI-

A'

x RI ix

LPI

sS

IpMSV-

'

XMLV Clone 61

8.8 kb Xho-RI

l-_8.8 kb al_x RI

,,,, .,,,,,.. ,,,,,,,,, XR

XMLV Clone 61

C

SU~~SRI

HD

p9-

pMSV-14.7 kb Sma -Hind m

FIG. 2. Substitution of M-MSV sequences into M-MuLVDNA. (A and A') Restriction maps of A-MLV clones 48 and61 (19). (B) Map for pMSV-1 (20). pMSV-1 is a clone of un-integrated M-MSV DNA in the HindIl site of pBR322. (C)Starting material for the cloning was pSLT, a subclone ofpMSV-1 resulting from deletion between the Sal I site in M-MSV DNA and the Sal I site in pBR322. Construction in-volved subcloning intopSLT ofthe 4.7-kbXhoI/Kpn I frag-ment of A-MLV clone 48 (p49) followed by removal of theXho I site by digestion withXho Iand nucleaseBAl-31 (p49-X-). The 4.7-kb Sma I/HindE fragment of pMSV-1 wasthen inserted. A complete proviral organization was regen-erated by inserting the 8.8-kb Xho I/EcoRI fragment fromA-MLV clone 61 (pMSV-MLV). The Sma I sites in the MSVand M-MuLV LTRs are equivalent, as are the unique XhoI sites. X, Xho I; K, Kpn I; Sm, Sma I; S, Sal I; HD, Hindu;RI, EcoRI.

glycosylated gag polyprotein (30). The complete sequence ofthe clone 124 strain of M-MSV has been determined (31), andthe region 5' to the initiation codon for Pr659'9 contains a num-ber of base changes, deletions, and substitutions. Some or oneof these alterations presumably cause the loss of glycosylatedgag polyprotein. The nucleotide sequences also indicate thatthe amino-terminal portions of M-MuLV Pr659'9 and M-MSVPr629'9 are highly conserved. Therefore, the 5' portion of a re-

combinant clone of M-MSV was introduced into a M-MuLVproviral organization between the Sma I site in the 5' long ter-minal repeat and the Xho I site at 1.558 kb (Fig. 2B). Theserestriction sites are equivalent for the two viruses. DNA fromthe resulting plasmid clone (pMSV-MLV) was then used totransfect NIH-3T3 cells.

Recovery of Infectious gPr8Om-Negative Virus. Transfec-tion of both of the recombinants described resulted in the pro-duction of infectious M-MuLV as measured by the UV-XC plaqueassay. For the Ab-MuLV recombinant, one XC plaque was ob-served in the second transfer of transfected cells and the cul-tures were uniformly positive for XC syncitia at the third trans-fer. The appearance of infectious virus only after the third passagemay have resulted from the fact that an in vitro ligation reactionwas transfected. The reaction mixture probably contained rel-atively low amounts of DNA ligated in the proper order for a

complete provirus. The resulting virus from the transfected cellswas termed Ab-X-MLV.

Transfection of cells with the pMSV-MLV plasmid also yieldedXC plaques on the second transfer, and the culture was uni-formly positive at the next transfer. Parallel transfection witha plasmid containing wild-type M-MuLV showed XC plaquesat the first transfer and confluent XC syncitia at the second.The relative delay in appearance of infectious virus in the pMSV-MLV transfection cannot be attributed to in vitro ligation andmay reflect a reduced rate for virus spread (see below).

Cells infected with either mutant virus were tested for syn-thesis of gag-related proteins. Cultures were labeled for 1 hrwith [3S]methionine, and gag-related products were immu-noprecipitated from cytoplasmic extracts by incubation withrabbit anti-p30 serum followed by harvesting of immunocom-plexes with fixed Staphylococcus aureus. p30 is the major gagprotein of the virus particle, and the anti-p30 serum has beenshown to efficiently immunoprecipitate gag polyproteins fromM-MuLV-infected cells (8). Immunoprecipitated proteins werereleased from the S. aureus and analyzed by NaDodSO4/poly-acrylamide slab gel electrophoresis followed by fixation, stain-ing, and fluorography of the gels (Fig. 3). For comparison, wild-type M-MuLV-infected NIH-3T3 cells (clone A9) were labeledand immunoprecipitated in parallel. Immunoprecipitation ofwild-type-infected cells showed gP80ag and Pr655ag (Fig. 3,lanes a and e). In addition, the gag-pol fusion precursor for re-

verse transcriptase (PrI809agsPol) was evident as a doublet. Dur-ing the labeling period, some processing of both gPr8Ogag andPr655ag occurred. In the case of Pr65" , processing resulted inmature p30 protein, while gPr8Ogag yielded polyproteins withadditional carbohydrate evident as diffusely migrating materialbetween 80 and 95 kDa. In contrast, cells infected with bothmutant viruses showed synthesis of Pr659ag and its subsequentprocessing to p30 but no synthesis of gPr8Ogag, as predicted fromthe constructions (Fig. 3, lanes c and g). A minor gag-relatedpolyprotein with an electrophoretic mobility similar to wild-typegPr80gag was evident in immunoprecipitates from cells infectedwith either mutant. However, this minor polypeptide repro-ducibly gave a slower mobility than gPr8Ogag; it may have alsobeen present in wild-type-infected cells but obscured by themuch larger amounts of gPr80gag. Cells infected with eithermutant also showed a somewhat reduced rate of processing ofPr659ag. This was evident as a lower p3O/Pr659a- ratio at theend of the 1-hr labeling period. In the mutant-infected cells,

A

XLR XR1- I RIRI

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A Ba b c d e f g h

- Pr 180 gg-Pol-

gPr8BO99Pr65930

p30 -~

FIG. 3. gag-Related proteins in infected cells. (A) Two 9-cm dishesofAb-X-MLV-infected cells and wild-type-infected cells (clone A9) werelabeled for 1 hr with [3S]methionine, and cytoplasmic extracts were

prepared. The extracts were divided in half, and one-half was treatedwith anti-p30 serum while the other-half was treated with normal rab-bit serum. The immunoprecipitates were harvested with fixed S. au-

reus and analyzed by NaDodSO4/polyacrylamide (12.5%) gel electro-phoresis. A fluorograph of the gel is shown. Lanes: a and b, extractsfrom wild-type-infected cells; c and d, extracts from Ab-X-MLV-in-fected cells; a and c, immunoprecipitation with anti-p30 serum; b andd, immunoprecipitation with normal serum. (B)An experiment similarto that ofA, except MSV-MLV-infected and wild-type-infected cells werecompared. Lanes: e and f, extracts from wild-type-infected cells; g andh, extracts from MSV-MLV-infected cells; e and g, immunoprecipita-tion with anti-p30 serum; f and h, immunoprecipitation with normalserum.

the higher molecular weight member of the Pr1809O9"P° dou-blet was also missing, supporting the previous conclusion thatit represents a fusion of glycosylated gag sequences to pol se-

quences. These results confirmed the construction of mutantM-MuLVs deficient in the ability to produce gPr8Ogag but stillable to produce Pr659'-. Furthermore, inability of the mutantsto produce gPr8Og"t did not abolish infectivity in tissue culture.

Characterization of Mutant Virus. Cells infected with thetwo mutant viruses were characterized with respect to the amountof virus produced (Table 1). Infectious virus production was

measured by the XC plaque assay, and virus-particle produc-tion was measured by reverse transcriptase activity. Cells in-fected with Ab-X-MuLV produced as much virus as wild-type-infected cells, by either infectivity or reverse transcriptase.Therefore, deletion of the glycosylated gag pathway does notaffect the rate of virus production from cells chronically in-fected with mutant virus. It should be noted that the particle/infectivity ratio of the mutant virus was also equal to that ofwild type.

In contrast, cells infected with MSV-MLV produced consid-erably less virus by either measure. MSV-MLV differed fromAb-X-MLV in the fraction of M-MuLV sequences substituted.For Ab-X-MLV, the only difference from wild-type M-MuLVwas confined to the 177-bp Pst I fragment. However, MSV-MLVcontained approximately 1.5 kb of MSV sequence, includingsubstitutions and deletions. The reduced virus production byMSV-MLV-infected cells probably resulted from sequence al-terations other than those leading to loss of gPr80g"g synthesis.

Differences in XC plaque morphology were observed for bothmutant viruses in comparison with wild type. For Ab-X-MLV,well-defined macroscopically visible XC plaques equal in sizeto wild-type M-MuLV were observed when optimal conditionswere used (XC cell overlay 5 days after infection). However,when XC cell overlay was done after 3-4 days, distinct XCplaques were observed on wild-type-infected cells but few couldbe detected on the Ab-X-MLV-infected cells. Microscopic ob-servation of the Ab-X-MLV-infected cells revealed areas ofsyncitial formation, but the degree of XC cell fusion was lessthan for the parallel wild-type-infected cultures. This suggestedthat early in infection the rate of initial virus release from mu-

Table 1. Virus production from NIH-3T3 cells infected withmutant and wild-type MuLV

Virus released

ReverseInfectivity,* transcriptase,t

Exp. Infecting virus XC pfu/ml cpm x 10-31 Wild-type M-MuLV 4.5 x 106 1,060

Ab-X-MLV 3.8 x 106 9902 Wild-type M-MuLV 1.2 x 106 10,750

MSV-MLV 3.8 x 104 822

Virus released into tissue culture supernatant from 106 infected cellsduring 24 hr was analyzed.* Infectivity oftissue culture supernatants on NIH-3T3 cells by the UV-XC plaque assay. pfu, Plaque-forming units.

tReverse transcriptase activity was measured in virus concentratedfrom 24-hr tissue culture supernatants. An exogenous reverse tran-scriptase assay with poly(rA)-oligo(dT) was used (16); values repre-sent calculated incorporation of [a-32P1dTTP in poly(dT) within 1 hrfrom 1 ml ofsupernatant. Actual values of incorporation ranged from20,000 to 400,000 cpm. Different preparations of [a-32P]dTTP wereused in experiments 1 and 2, so incorporation values can only be com-pared within each experiment.

tant-infected cells or the rate of viral envelope glycoprotein ap-pearance (responsible for XC cell fusion) is reduced in com-parison with that of wild-type-infected cells. Infection of cellswith MSV-MLV showed a greater difference in XC plaquemorphology. Even under optimal conditions, XC plaques fromthe mutant virus, while distinct, were smaller than the plaquesfor wild-type-infected cells. This would be expected from thereduced rate of virus production in the MSV-MLV mutant.

Mutant virus showed another difference from wild type. Theresults when recently produced virus was harvested from Ab-X-MLV- and wild-type-infected cells at 2-hr intervals after a 1-hr labeling period with [3S]methionine and analyzed by band-ing to equilibrium density in sucrose gradients are shown inFig. 4A. Virus from the Ab-X-MLV-infected cells had a higherdensity (1.185 g/ml) than virus from wild-type-infected cells(1.170 g/ml). This suggested that the physical nature of virusfrom mutant-infected cells differed somewhat from that of wild-type virus. However, any difference in physical structure wasminor; NaDodSO4/polyacrylamide gel electrophoresis of totalmethionine-labeled viral proteins (Fig. 4B) did not reveal anydifferences between mutant and wild-type virus.

DISCUSSIONThe construction of M-MuLV mutants defective for gPr809agsynthesis but able to synthesize Pr65sag is described here. Theresults indicate that the glycosylated gag pathway is not ab-solutely required by M-MuLV for productive infection in tissueculture. However, minor effects on virus replication were sug-gested by the altered XC plaque morphology of both mutants.The glycosylated gag pathway might provide some accessoryfunction in virus replication. The data in Fig. 3 suggest that lackof this pathway may have a minor effect on virus maturation.Cleavage of Pr655ag to the mature internal structural proteinsoccurs as a late step during virus maturation, concomitant withbudding and release from the cell (2). The reduced rate of Pr659`9processing in the mutant-infected cells may reflect a longermaturation time for mutant virus. Cells chronically infected withmutant virus, however, produced essentially the same amountas cells infected with wild-type M-MuLV (Table 1).An interesting property of the mutant virus is the slightly

higher buoyant density, presumably reflecting an altered phys-ical structure. One possible explanation is release of immaturevirus from mutant-infected cells. However, NaDodSO4/poly-

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Ba b c

-Pr 180g'g-P°gPr80690

gp 70 _ u I- Pr 659:

gp45-£-

p3 - - p30

p15E

p12E -

FIG. 4. Density of virus from mutant- and wild-type-infected cells.(a) Four 9-cm dishes of wild-type- and Ab-X-MLV-infected cells werelabeled for 1 hr with [5Slmethionine as in Fig. 3. Tissue culture su-pernatants were harvested from the cells at 2-hr intervals and storedin ice. The supernatants were clarified by low-speed centrifugation, andvirus was harvested by high speed centrifugation. Resuspended viruswas layered onto linear 25-55% (wt/vol) sucrose gradients and sedi-mented to equilibrium density by centrifugation overnight in a Beck-manSW 50.1 rotor at 35,000 rpm. The gradients were fractionated, andthe radioactivity in equal portions of each gradient fraction was mea-sured by scintillation counting. Virus harvested between 2 and 4 hr afterlabeling is shown, with the results from two gradients superimposed.*, Ab-X-MLV virus; o, wild-type virus. x, Buoyant density, as deter-mined from the refractive index; the density distributions for the twogradients were the same. (b) Labeled virus harvested at 2-hr intervalsfrom Ab-X-MLV- and wild-type-infected cells was pooled from peakgradient fractions, diluted with buffer, and pelleted by centrifugationin an SW 50.1 rotor at 35,000 rpm. Virus in the pellets was then ana-lyzed by NaDodSO4/polyacrylamide gel electrophoresis. For compar-ison, gag-specific proteins from wild-type-infected cell cytoplasm im-munoprecipitated with anti-p30 serum were also analyzed (lane c). Lanea, virus from wild-type-infected cells; lane b, virus from wild-type Ab-X-MLV-infected cells. The positions of the mature viral proteins areindicated. Two of the mature gag proteins, p15 and p1O, are not evidentin the autoradioagram because they lack methionine (27).

acrylamide gel electrophoresis of mutant virus revealed normalpatterns of viral proteins (Fig. 4B). Labeled virus from the mu-tant-infected cells was also examined for increased levels of un-cleaved gag polyprotein precursors by immunoprecipitation with

anti-p30 serum, but no increased levels were detected.The glycosylated gag pathway is encoded by all replication-

competent strains of MuLV and by all strains of feline leukemiavirus as well (2). This suggests that this pathway has been con-served during viral evolution, even though it is not required forreplication in tissue culture. It may be important for infection,replication, or pathogenesis in the natural host.

We thank Owen Witte for communicating results prior to publicationand for providing materials. We also thank Inder Verma for materials.This work was supported by Grants CA32455 and CA32454 from theNational Cancer Institute. M.F. was supported by Training GrantGM07311 from the National Institutes of Health.

1. Coffin, J. (1982) in Molecular Biology of Tumor Viruses: RNA Tu-mor Viruses, eds. Weiss, R., Teich, N., Varmus, H. & Coffin, J.(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), 2ndEd., pp. 261-368.

2. Dickson, C., Eisenman, R., Fan, H., Hunter, E. & Teich, N. (1982)in Molecular Biology of Tumor Viruses: RNA Tumor Viruses, eds.Weiss, R., Teich, N., Varmus, H. & Coffin, J. (Cold Spring Har-bor Laboratory, Cold Spring Harbor, NY), 2nd Ed., pp. 513-648.

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255-278.10. Evans, L. H., Dresler, S. & Kabat, D. (1977)1. Virol. 24, 865-

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Genetics: Fan et al.

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