Vol. 66, No. 2

JOURNAL OF VIROLOGY, Feb. 1992, p. 946-955

0022-538X/92/020946-10$02.00/0 Copyright X) 1992, American Society for Microbiology

gag as Well as myc Sequences Contribute to the Transforming Phenotype of the Avian Retrovirus FH3 ANDREI T. TIKHONENKO AND MAXINE L. LINIAL* Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104-2092 Received 9 September 1991/Accepted 4 November 1991

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline-*histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.

tion by MC29, OK10, and MH2 (1, 22, 26). Moreover, chicken c-myc, when activated in B cells by proviral insertion, also contains several amino acid changes, including the amino acid change at position 61 (59). However, as the genome of another myc retrovirus, CMII, contains only a single mutation in the last myc codon (57), it was concluded that no specific mutations are required for myc activation. To further analyze the significance of v-myc mutations, different c-myc-containing retroviruses have been constructed. It was found that viruses containing either the c-myc or the gag-c-myc oncogene do not induce complete fibroblast transformation in vitro, as measured by focus assay and cell morphology (14, 19). However, macrophages can be transformed even by nonmutated c-myc (19, 55). These studies argue that c-myc potentiation is a tissuespecific phenomenon: in fibroblasts, it requires both overexpression and point mutations, while in macrophages, overexpression alone is sufficient for transformation. In contrast, a mutant of MC29 that fails to transform chicken macrophages yet still is able to transform quail macrophages and fibroblasts has been described (3). These data suggest than oncogene activation can be also a species-specific phenomenon. Analysis of the contribution of specific amino acid changes to v-Myc-induced transformation is complicated by the fact that in the best-studied v-myc virus, MC29, as well as in CMII, v-Myc is expressed as a Gag-Myc fusion protein. To elucidate the possible role of the Gag portion in transformation, it was deleted from the MC29 genome. This deletion did not change the transforming ability of the encoded virus (49). In addition, the avian retrovirus OK10 (22) expresses Myc without a retroviral fusion partner, as does at least one naturally occurring feline retrovirus, FTT (16). Thus, it has been argued that Gag sequences simply serve as a carrier

Retroviral transduction is one means by which cellular proto-oncogenes can be activated. Acquisition of cellular sequences gave rise to the majority of acutely transforming retroviruses capable of inducing sarcomas, leukemias, and carcinomas. Capture of a cellular proto-oncogene by a retroviral genome usually leads to (i) elevation of the level of oncogene expression and (ii) mutations in the nucleotide sequence (for a review, see reference 5). Extensive studies suggest that both mechanisms can lead to proto-oncogene potentiation, although in most cases both events occur and their specific contributions remain to be elucidated (5). One of the best experimental models to address this issue is the cellular proto-oncogene myc. Several avian (MC29, CMII, OKIO, MH2, and FH3) and feline (myc feline leukemia viruses) myc-containing retroviruses have been isolated (5, 11, 33, 39, 40). All of the v-myc genes described so far are highly homologous to their cellular counterparts, but the v-Myc proteins always differ by at least one amino acid from c-Myc. The specific amino acid changes tend to be similar in independently derived v-myc viruses; for example, MC29, OK10, and MH2 each contain a mutation which alters amino acid 61 (1, 9, 22, 57). The absence of v-myc genes identical to c-myc has raised the possibility that specific mutations in coding sequences are required for myc activation. It has been demonstrated that substitution of v-myc sequences by c-myc significantly decreases the ability of the resultant virus to transform chicken embryo fibroblasts (CEFs). Conversely, the single threonine-to-methionine change at position 61 confers transforming potential to c-Myc (19). Substitution of one of the hydrophilic amino acids at positions 325 to 335 by a hydrophobic amino acid is also considered to be significant because this change accompanies viral transduc*

Corresponding author. 946

VOL. 66, 1992

TRANSFORMING ACTIVITY OF GAG-MYC FUSION PROTEINS

and are not important for transformation by MC29 and related viruses (6, 55). Nevertheless, it is still conceivable that the Gag portion could grant the oncoprotein some new functions and thus affect its transforming activity. Until recently, it was difficult to address this question because the specific function of the Myc protein itself was unknown. Indirect evidence suggested that Myc might be involved in cell cycle control or gene regulation (13, 37). Very recently, a nucleotide sequence for specific Myc binding has been determined (7, 45), and a protein which heterodimerizes with Myc, called Max, has been discovered (8, 44). The interaction of Myc and Max involves helix-loop-helix motifs in the proteins, and the Myc-Max heterodimer has higher affinity for an in vitrodefined Myc binding sequence than does Myc alone (8). It has also been shown that in vivo Myc is involved in either activation (17, 27) or repression (60) of specific genes. These findings allow Myc to be classified as a member of a family of transcription factors which contain two DNA-binding motifs: basic region-helix-loop-helix and basic region-leucine zipper. Although the relationship of Myc function to transformation is unclear, this question can be addressed by studying activated Myc proteins with different transforming

potentials.

We have been examining the genomic structure of a new avian myc-containing retrovirus, FH3 (11), which, like MC29, CMII, and myc feline leukemia virus (4, 9, 23, 46), encodes a Gag-Myc fusion protein. However, FH3 is unique in that it has retained almost the entire gag gene, lacking only the carboxy-terminal 4 kDa. The gag gene of avian retroviruses encodes a polyprotein, Pr769'1% which is processed into five proteins which are (from the amino terminus) matrix (MA), a 10-kDa protein of unknown function (plO or X), capsid (CA), nucleocapsid (NC), and protease (PR) (15). The Gag protein of CMII contains only MA and part of plO, and that of MC29 contains MA, plO, and part of CA, whereas FH3 encodes MA, p10, CA, NC, and part of PR. In fact, FH3 is the only described oncogenic retrovirus encoding a Gag-Onc fusion protein with capsid and nucleocapsid determinants. These determinants are responsible for virion protein multimerization and RNA binding, respectively (30). It is likely that additional functional domains might confer to the FH3 Gag-Myc oncoprotein some unusual features. Indeed, FH3 is distinguished from the other members of the MC29 family because it possesses only limited transforming activity and fails to acutely transform chicken or quail fibroblasts as measured by focus assay. A similar phenotype has been cdescribed for an MC29 mutant with a C-terminal deletion in Myc (18). However, our previous data demonstrated that FH3 Myc does not contain any extensive deletions, confirmed here by further sequence analysis. Thus, it seemed possible that in FH3, extended Gag region plays a role in the transforming abilities of Myc. Data presented in this report demonstrate that sequences within the carboxy half of FH3 Gag suppress the fibroblast-transforming activity of v-Myc. Moreover, during in vitro selection for fibroblast transformation, variants which have deleted this portion of Gag emerge, suggesting the significance of Gag determinants for oncogenicity. MATERIALS AND METHODS Viruses and tissue culture. The original isolate of FH3 (FH3E) has been described previously (11). A late variant (FH3L) capable of transforming fibroblasts appeared after

947

propagation of FH3E for several months in quail embryo fibroblasts (QEFs). We picked several foci after 21 cell passages and established mass culture of transformed virusproducing cells. Molecular clones of FH3E and FH3L as well as of MC29 were transfected into QEFs by a modified calcium phosphate precipitation method (12). To obtain replication-competent stocks, helper virus RCAS/BP DNA (43) was cotransfected with provirus-containing plasmids, and viruses were harvested 10 days after transfection. Both QEFs and CEFs were grown in GM medium (34) supplemented with 1% dimethyl sulfoxide and 1% heat-inactivated chick serum. Chicken macrophages were isolated from whole blood by centrifugation in Ficoll (Pharmacia) gradients and grown in CM medium as previously described (34). For fibroblast focus assays, CEFs and QEFs were trypsinized and 5 x 105 cells were seeded onto 35-mm plates. Infections were performed overnight in the presence of Polybrene (2 ,ug/ml). Twenty-four hours after infection, the cells were overlayed with GM-1% dimethyl sulfoxide containing 0.72% agar and after another 48 h reoverlayed with GM containing 0.8% agar. For macrophage focus assays, 107 peripheral blood cells were seeded onto 35-mm plates and infected 72 h after plating and removal of nonadherent cells. The overlay recipe was as follows: 2x GM, 22.2 ml; CM, 17.8 ml; and 1.8% agar, 15.0 ml. Foci were counted 10 to 14 days after infection. For growth in soft agar, several dilutions (typically 105, 104, and 103 cells per 60-mm plate) of infected and uninfected QEFs were plated in soft agar as previously described (34). Radioimmunoprecipitation. Subconfluent cells were washed thoroughly, labelled for 45 min with [35S]methionine (0.25 mCi/100-mm plate), and rewashed twice with ice-cold phosphate-buffered saline (PBS). Cells were disrupted in Ab buffer (21) (high-stringency conditions), sonicated for 30 s, and centrifuged at 10,000 x g for 15 min. In the case of low-stringency radioimmunoprecipitation, cells were disrupted in PBS with 1% Nonidet P-40 (8) instead of Ab buffer. Cellular extracts were then incubated for 90 min with protein A linked to Sepharose 4B beads (Pharmacia) and either anti-v-Myc (21) or anti-Max (8) antibodies. Precipitated proteins were separated in 10% denaturing polyacrylamide gels and visualized by autoradiography. PCR amplification, molecular cloning, and DNA sequencing. To obtain the molecular clones of FH3 variants, we collected viral particles from infected cultures and clarified them by low-speed centrifugation. Viral particles were then pelleted through a 20% sucrose cushion, treated with RNasefree DNase at 37°C for 15 min, and disrupted by addition of sodium dodecyl sulfate (SDS) to 0.5%. Viral RNAs were phenol-chloroform extracted, ethanol precipitated, dissolved in RNase-free water, and used for cDNA synthesis with random hexamers followed by polymerase chain reaction (PCR) amplification (28). PCR primers were designed such that they encompassed the gag-myc coding region and contained restriction enzyme sites (Fig. 1). The nucleotide sequences of PCR oligonucleotides were as follows: 5'-TT GAGTGGTCAGATCTCCCGCCTT-3'(BGL), 5'-GCATGG GAATTCTCACTCCTATCC-3' (ECA), 5'-GATAGGAGT GAGAATTCCCATGCG-3'(ECS), 5'-ACTGGTACCCCTA TGCACGAGAGT-3' (KPN), and 5'-TTATAGCGGCAGC CACTCGCGACC-3 (NRU). In the case of the KPN primer, a KpnI restriction site was added to its 5' end for cloning purposes. PCR reactions were performed in the presence of 10% glycerol, and for each of 30 cycles, the extension time was 8 min. These conditions are known to facilitate the amplification of long (-2 kb) and GC-rich DNA sequences

J. VIROL.

TIKHONENKO AND LINIAL

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A

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gag

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enzymes and

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pRCAS/BP

Retroviral vector

FIG. 1. Strategy for the molecular cloning of FH3E (A), MC29 (B), and FH3L (C). Dashed lines indicate internal gag deletion in the FH3L Arrows indicate primers used for PCR amplification. Prior to insertion into RCAS/BP, NruI-EcoRI and EcoRI-KpnI PCR fragments of FH3L were ligated in pSL1180. Since the NruI site is not unique in pRCAS/BP, the Xhol-NruI restriction fragment was removed from RCAS/BP gag and ligated to a reconstituted NruI-KpnI gag-myc fragment. The resultant XhoI-KpnI fragment was reassembled into RCAS/BP so that the gag gene sequence was not interrupted. For other details, see Materials and Methods.

genome.

while having no effect on the amplification of non-GC-rich sequences (51; data not shown). PCR products were gel purified, cut with the appropriate restriction enzymes, and subcloned either into pSL1180 (Pharmacia) (10) or directly into pRCAS/BP (43) so that the gag-pol fusion gene was substituted by the gag-myc fragment of interest. For control PCR reactions, we used a plasmid containing the neo gene (35) and an MC29 proviral clone (56), in nonpermuted form (29), inserted into the pVZ1 vector (32). DNA sequencing was performed on double-stranded templates by the method of Sanger et al., (48) using Sequenase kits (United States Biochemical). Nucleotide sequence accession number. The GenBank accession number for the FH3 myc gene is M74581. RESULTS Molecular cloning and nucleotide sequence analysis of the original FH3 isolate myc gene. In our previous report (11), we described the partial nucleotide sequence of several cDNA clones corresponding to the FH3 genomic RNA. These data

demonstrated that the myc gene of FH3 is linked to the gag sequences and is expressed as a 145-kDa Gag-Myc fusion. To obtain a molecular clone with the phenotype of the original isolate (FH3E), we used PCR to amplify the FH3 gag-myc region. Insertion of the amplified fragment into a retroviral molecular clone would give rise to a viral construct which could, in the presence of helper virus DNA, initiate productive infection and transformation. The retroviral vector pRCAS/BP used in these studies contains two convenient restriction sites, BglII and KpnI, which lie within the capsid portion of gag and just upstream of the env splice acceptor site, respectively (43). Hence, neither the gag nor the env gene of the vector would be disrupted by substituting of the pRCAS/BP BglII-KpnI fragment with a PCR gag-myc fragment bounded by BgiII and KpnI sites; however, the pol sequences would be completely deleted (Fig. 1). We designed two PCR primers: a 5' oligonucleotide (BGL) homologous to the region encompassing the BgiII site in the Rous sarcoma virus (RSV) gag and a 3' oligonucleotide (KPN) containing the myc termination codon and an added KpnI restriction site at its 5' end. The same pair of primers was

TRANSFORMING ACTIVITY OF GAG-MYC FUSION PROTEINS

VOL. 66, 1992 TABLE 1. In vitro transformation assays

1 2

FFU/ml" Expt

I

II

III

IV

Virus

RCAS/BP FH3E RCAS/FH3E RCAS/FC MC29 RCAS/MC FH3L RCAS/FH3L RCAS/FH3E RCAS/EL RCAS/FH3L RCAS/LE RCAS/MC RCAS/RM

949

200 K

FH3E Pt-14

Chicken

macrophages