VIROLOGY

99, 358-3’71 (1979)

Gene Order of Mouse Mammary Tumor Virus Precursor Polyproteins Their Interaction Leading to the Formation of a Virus’

and

RICHARD J. MASSEY2 AND GERALD SCHOCHETMAN Biological

Carcinogenesis

Program,

Frederick

Cancer Research Center, Frederick,

Accepted August 26,

Maryland

21701

1979

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75”“” containing gp52 and gp36, and Pr750ugcontaining ~27, pp20, ~14, and ~10. The gene order for gPr75’“” has been previously shown to be H,N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75”“” is H,N-plO-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain ~14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [Wr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of “Wabeled MMTV cell surface antigens by immune precipitation with MMTV antigp52, gp36, ~27, ~14, and p10 sera followed by SDS-PAGE revealed only lesI-gp52. In contrast, cell surface glycoprotein labeling revealed [“H]gp52 and 13H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75Y”* and gp52 but not gPr75’““. Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr750agmolecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule. INTRODUCTION

The mouse mammary tumor virus (MMTV) contains seven proteins with molecular weights of 10,000 (plO), 14,000 (p14), 20,000 (pp20), 27,000 (p27), 30,000 (p30), 36,000 (gp36), and 52,000 (gp52) daltons (Table 1). gp52 and gp36 are the major virion glycoproteins and gp52 is the major external protein of the virus (Cardiff et al., 1974, 1978). p10 is a hydrophobic protein (Cardiff et al., 1978; Marcus et al., 1978) and a viral * The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 2 To whom reprint requests should be addressed. 358

membrane-associated protein (Cardiff et al., 1978), whereas ~27 and p14 are associated with the virus core (Teramoto et al., 1977). Further studies have demonstrated that p14 is a DNA binding protein that represents the virus ribonucleoprotein (Longet al., 1977)and that pp20 is the major virion phosphoprotein (Racevskis and Sarkar, 1979). It has been suggested that p30 may be an intermediate cleavage product containing p14 (Gautsch et al., 1978). The virus also contains an RNA-dependent DNA polymerase (RDDP) (Marcus et al., 1976). Previous studies have demonstrated that these MMTV structural proteins are independently synthesized (Schochetman and Schlom, 1976) as two distinct precursor

MMTV PRECURSOR POLYPROTEINS

359

TABLE 1

PROPERTIESOFMMTVSTRUCTURALPROTEINS Genetic locusa

Designation (MW)b

gag r--P?75 Pi-1604 I -E PO1

r--------L----W---

env

gpr’“-Cgp36

p10 (10,000) p14 (14,000) pp20 (20,000)

Location in virus

Properties

p30 (30,000) RDDP (?)

Membrane Core Core Core Core Core

Hydrophobic protein Ribonucleoprotein (ssDNA binding protein) Major phosphoprotein Major group-specific internal antigen Possible intermediate cleavage product RNA-dependent DNA polymerase

gp52 &VW (36,000)

Envelope Envelope

Major external virion glycoprotein Major envelope glycoprotein

$7 (27&W

a Individual virus genetic loci designated as previously proposed (Baltimore, 1974). * Molecular weight determinations are based on SDS-PAGE.

polyproteins (Dickson et al., 1976; Schochetman et al., 1977, 1978c; Dickson and Atterwill, 1978; Nusse et al., 1978; Racevskis and Sarkar, 1978). One contains the virion glycoproteins gp52 and gp36 (gPr75”““), and the other contains the virion nonglycoproteins ~27, pp20, ~14, and p10 (Pr759a9)(see Table 1). GPr75”“” is cleaved intracellularly (Dickson et al., 1976; Schochetman et al., 1977) leading to the appearance of gp52 on the cell surface as demonstrated by lactoperoxidase-catalyzed iodination of cell surface proteins (Schochetman et al., 1978a, b). Presumably, gp36 is also integrated into the cell membrane based on its location in extracellular virus (Cardiff et al., 1974; Sarkar et al., 1976). In contrast, Pr75gaQis independently assembled in the cytoplasm into intracytoplasmic A particles which are precursors to MMTV cores (Sarkar and Whittington, 1977;Tanaka, 1977; Arthur et al., 1978; Cardiff et al., 1978), and Pr75gagremains in an uncleaved form in the Mm5mt/c, C3H mammary tumor cell (Schochetman et al., 1978c). However, in certain cells Pr75g”gundergoes intracellular processing (Dickson and Atterwill, 1978; Dahl and Dickson, 1979) yielding intermediate gag cleavage products. The presence of these intermediate gag products together with the development of antisera directed against individual MMTV proteins have enabled us to define the MMTV gag gene order.

The assembly of a virus particle requires the interaction of an intracytoplasmic A particle with a membrane site containing gp52 and gp36 leading to the budding of a virus particle. This interaction would be mediated by one or more sites on Pr750ag which represent different internal virus structural proteins upon cleavage of the polyprotein. Thus, knowledge of the localization of MMTV proteins in the cell membrane of a mammary tumor cell would provide an understanding of the assembly of a virus particle. Therefore, the cellular localization of MMTV proteins was studied using (i) lactoperoxidase- and galactose oxidase-catalyzed radiolabeling of cell surface antigens, (ii) membrane immunofluorescence, and (iii) complement-dependent serum cytotoxicity. These techniques were applied to cells treated with EDTA to modify cellular membranes so as to expose MMTV antigens buried in the membrane. MATERIALS

AND METHODS

Cells. The Mm5mt/c, cell line was originally derived from C3H mouse mammary tumor cell cultures and developed into a continuous cell line producing high levels of MMTV (Owens and Hackett, 1972; Fine et al., 1974). The 13D cells are a single cell clonal isolate of Mm5mt/c, cells (Schochetman et al., 1978b). GR/N mouse mammary

360

MASSEYANDSCHOCHETMAN

tumor cells were established in our labora- with EBSS and lysed in situ with lysis buffer (0.01 M Tris-HCl, pH 8.0, 0.15 M NaCl, tory as previously described (Arthur et al., 0.001 M EDTA, 1% Triton X-100, and 1978). CrFK feline cells infected with MMTV from C3H mice (Howard et al., 1977) were 0.5% DOC). Radioactive labeling of intracellular viral kindly supplied by Dr. J. Schlom (National Cancer Institute, Bethesda, Md.). The C3W polyproteins. Cell cultures (80% confluent) lOT1/2 cell line was derived from a C3H were incubated for 2 hr in leucine-deficient medium containing 10% dialyzed fetal calf mouse embryo and did not produce MMTV or contain MMTV antigens. All cells were serum. The cells were then pulse-labeled propagated in Dulbecco’s modified Eagle’s for.10 min with [3H]leucine (40 Ci/mmol) at a medium containing 10% fetal calf serum final concentration of 50 pCi/ml. After incu(DMEM) and supplemented with dexametha- bation, the cells were washed twice with cold phosphate-buffered saline, lysed in situ, sone (10 pg/ml) and insulin (10 Fg/ml). Antisera. Rabbit antisera directed against and clarified prior to use for immunopreMMTV and antisera directed against the cipitation (Schochetman et al., 1978c). Radioimmunoprecipitation and gel elecMMTV proteins gp52, gp36, ~27, ~14, and p10 were prepared and characterized as trophoresis. Lysates of surface-labeled and described earlier (Arthur and Fine, 1979). internally labeled cells were clarified by The specificity of each antiserum was evalu- centrifugation and 150~~1 aliquots were ated by titrating against lz51-labeledpurified incubated with 50 ~1 of primary viral antiMMTV proteins as previously described sera for 1 hr at 37” and then overnight at (Arthur et al., 1978). 4”. The immune precipitates were collected, washed, and prepared for SDS-PAGE on Radiolabeling of cell surface antigens. Cells were seeded into tissue culture flasks 5-20% gradient slab gels as previously de48 to 72 hr prior to radiolabeling. Cell mono- scribed (Schochetman et al., 1978c). The layers (90% confluent) were surface labeled lz51-labeled proteins were visualized by using the lactoperoxidase-catalyzed iodina- autoradiography and 3H-labeled proteins tion technique as previously described (Witte and glycoproteins by fluorography (Laskey and Weissman, 1976; Schochetman et al., and Mills, 1975). Radiolabeled, disrupted 1978b). Glycoproteins were surface labeled MMTV was coelectrophoresed as a marker by the galactose oxidase-sodium [3H]boro- for gp52. hydride method (Gahmberg and Hakomori, Single-stranded DNA afjkity chromatog1973). By this method terminal galactosyl raphy. [3H]Leucine-labeled (C3H-MMTV) or N-acetylgalactosaminyl residues are CrFK cell lysate was subjected to chrooxidized to the corresponding C-6 aldehydes matography on a single-stranded DNA by the enzyme galactose oxidase. The re- (ssDNA)-Sepharose column as previously sulting aldehydes are then reduced under described (Schochetman et al., 1978c). physiological conditions with tritiated boroComplement-&pen&nt serum cytotoxicity hydride. The reaction is potentiated by and membrane immwhoJuorescence assays. neuraminidase which removes terminal sialic Rabbit antisera to purified MMTV glycoacid residues. Briefly, the cells (in T-150 proteins and nonglycoproteins were tested cm2 flasks) were washed with Earle’s bal- in a complement-dependent serum cytotoxicanced salt solution (EBSS) without phenol ity (CDSC) assay based on 51Crrelease (Johnred. Neuraminidase, 125 units, and 25 units son et al., 1972). YZr-Labeled Mm5mt/c, of galactose oxidase in 5 ml of EBSS were target cells (104)were reacted in a microtiter added to the flask which was then incubated well with 0.05 ml of the heat-inactivated for 30 min at 37”. The cells were then washed antisera and 0.05 ml of rabbit serum diluted three times with EBSS and reduced for 30 1:5 as the complement source. This mixture min at room temperature with 2.5 mCi/5 ml was incubated at 37” for 5 hr, then 0.1 ml of NaB[3H], (7.2 Ctimmol). After labeling supernatant was removed and the amount by the lactoperoxidase-catalyzed iodination of [“‘Cr] released was measured in a Searle and the galactose oxidase-sodium [3H]boro- gamma counter. The percentage cytotoxicity hydride method, the cells were then washed was determined according to the formula:

MMTV

percentage cytotoxicity

=

PRECURSOR

361

cpm for test release - cpm for spontaneous release x 100. cpm for maximal release - cpm for spontaneous release

The test release was measured with target cells incubated in antisera and complement. The spontaneous release was determined for cells incubated in medium only, and the maximal release was determined by lysing the cells with distilled water and freezethawing three times. The cytotoxicity of the complement was tested in each assay by incubating the target cells in complement without the addition of any antisera. The percentage cytotoxicity with complement alone was always less than 10%. The immunofluorescence reaction was performed in two steps. First, lo6 target cells were pelleted and resuspended in 100 ~1 of rabbit antisera to purified MMTV glycoproteins and nonglycoproteins and incubated for 45 min at 37”. The cells were then washed by centrifugation through a l-ml cushion of fetal calf serum. Second, the pelleted cells were resuspended in 100 ,ul of 1:lO diluted fluorescein-conjugated goat anti-rabbit IgG serum (Hyland Laboratories) and incubated for an additional 45 min at 22”. The cells were then washed again by centrifugation through a l-ml cushion of fetal calf serum, and the final cell pellets were resuspended in 0.5 ml of Dulbecco’s phosphate-buffered saline for immunofluorescence analysis. RESULTS

Intracellular

POLYPROTEINS

MMTV Precursor Polyp-oteins

The gene order of the MMTV gag precursor polyprotein could be determined by antigenic analysis of intermediate cleavage products of the polyprotein. This required the use of highly characterized antisera directed against the MMTV proteins, gp52, gp36, ~27, ~14, and ~10. Figure 1 shows that each antiserum precipitated its homologous protein from iodinated MMTV. The 16,000dalton protein precipitated by the anti-p27 serum has been shown by tryptic peptide fingerprinting to be a fragment of p27 (Arthur et al., 1979). The specificity of the antisera for the individual MMTV proteins was further substantiated by endpoint titration of the individual antisera with lz51-labeled purified

MMTV proteins (Table 2). Each antiserum was only capable of precipitating its homologous protein. To study intracellular MMTV proteins, uninfected CrFK, (C3H-MMTV), CrFK, and GR mammary tumor cells pulsed for 10 min with [3H]leucine were lysed and virus-specific proteins were analyzed by SDS-PAGE following immunoprecipitation with the individual antisera (Figs. 2 and 3). The results show that the antisera precipitated a variety of proteins in the (C3H-MMTV) CrFK (Fig. 2A) and GR cells (Fig. 3) both of which were actively producing MMTV. In contrast, no detectable labeled proteins were precipitated from the uninfected CrFK cells (Fig. 2B) with any of the antisera demonstrating that the proteins observed in the infected cells were MMTV coded. The protein profile in the (C3H-MMTV) CrFK and GR cells were similar with some differences observed. The 75,000-dalton polyprotein precipitated by both anti-gp52 and anti-gp36 sera has been shown to be the precursor polypeptide to these two molecules and is termed gPr75”““. Anti-p27, anti-p14, and anti-p10 sera precipitated a number of precursor polyprotein cleavage products (see Table 3 for summary of precipitations). All three antisera precipitated a 160,000 (Pr160Q”Q)-, a 100,000 (PrlOOQ”Q)-,and a 75,000 (Pr75QQg)-daltonprotein; in the GR cell Pr75Q”Qwas slightly larger, about 77,000 daltons (Pr770ag). Four additional polypeptides were precipitated by these antisera. A protein of 38,000 (Pr380ag)was precipitated by only anti-p14 and anti-p27 sera, indicating that p27 and p14 were adjacent to each other. To further demonstrate that Pr38gaQcontained ~14, we utilized the observation that p14 binds specifically to ssDNA and that any protein containing p14 would also have a high affinity for ssDNA (Schochetman et al., 1978c; Long et al., 1977). Thus, [3H]leucine-labeled (C3H-MMTV) CrFK lysate was subjected to ssDNA-Sepharose affinity chromatography. The proteins were eluted by the stepwise addition of buffers contain-

MASSEY AND SCHOCHETMAN

362

ing 0.05, 0.15, and 0.6 M NaCl. The radioactive eldtion profile is shown in Fig. 4. Approximately, 18% of the total radioactivity was strongly bound to the column and could be eluted with 0.6 M NaCl. The radioactive fractions eluted at 0.6 M NaCl were pooled, dialyzed, and concentrated. Aliquots containing equal amounts of radioactivity were incubated with anti-p27, anti-p14, and anti~10 sera and the immune precipitates analyzed by SDS-PAGE. The results are presented in Fig. 4. As expected, only those proteins which were precipitated by the antip14 sera bound to the ssDNA confirming the presence of p14 on these intermediate cleavage products. The absence of Pr160 and Pr130 in the 0.6 M eluate was due to their poor recovery from the column. A protein of about 65,000 (Pr65ga”) which did not bind to ssDNA (Fig. 4) was precipitated by the anti-p27 and anti-p10 sera but not by the anti-p14 serum (Table 3) indicating that p14 must be located at one end of the PrWyUgmolecule. A protein of about 130,000 (Prl30Q”“) was precipitated by the anti-p27

TABLE 2 SPECIFICITY OF ANTISERA TO MMTV PROTEINS BASED ON RADIOIMMUNOPRECIPITATION OF lz51-L~BELED PROTEINS

‘Z51-Labeled proteins Antisera to

gp52

gp36

P27

P14

PlO

g~52 gP36 P27 P14 PlO

6400”