It is widely assumed that MMTV induces tumors by acting as an insertional mutagen that activates the expression of cellular protooncogenes. However, the median latency of mammary tumor formation in female mice transgenic for the Wnt1
oncogene was 5 months of age, with >80% of mice developing tumors by 7 months (55
). In addition, transgenic females rarely developed >1 tumor per mouse and never developed >3 tumors per mouse (55
). This relatively long latency to tumor development and the stochastic nature of mammary tumors in Wnt1
transgenic mice argues that Wnt1
contributes to, but is not sufficient for, tumorigenesis in these mice. Therefore, events in addition to oncogene expression are necessary for mammary tumor development. These experiments demonstrate unequivocally that the region of the MMTV Gag protein that differs between MMTV(C3H) and Mtv1
is involved in mammary tumorigenesis, since replacing the polymorphic region in Mtv1
Gag with that from MMTV(C3H) Gag was sufficient to convert the tumor-attenuated hybrid MMTV into a virus with high tumorigenic potential (Fig. ).
Whereas virus A has most of its sequences derived from tumorigenic MMTV(C3H) gag, including pp21, p3, p8, CA, and part of NC (CA/NC), virus C and virus B have CA/NC and pp21, p3, and p8 MMTV(C3H) gag sequences, respectively (Fig. ). Unlike virus A, which induces mammary tumors in 64% of infected C3H/HeN females by 250 days [similar to wild-type MMTV(C3H)], virus C induces tumors in only 31% of infected C3H/HeN females, and less than 10% of the mice succumbed to tumors when infected with virus B within the same period of time. Even though virus C does not induce mammary tumors with the same frequency as virus A [or MMTV(C3H)], the latency of virus C-induced tumors appears to be similar to the latencies of tumors induced by virus A and MMTV(C3H) (Fig. ). Two conclusions can be drawn from these results. First, the determinants of the tumorigenic capacity of the virus lie within the CA/NC sequences. Second, the pp21, p3, and p8 sequences are not required for tumor induction. However, it remains possible that the pp21, p3, and p8 sequences, together with CA/NC sequences, might accelerate gag-dependent tumorigenicity.
How can we explain the low frequency of mammary tumors in C3H/HeN mice infected with tumor-attenuated viruses B, D, and HP (Fig. )? Our working hypothesis suggests that Gag accelerates mammary tumor development by cooperating with cellular protooncogenes, as tumors with low incidence arise in MMTV-free transgenic mice constitutively expressing protooncogenes in the mammary glands (55
). Therefore, spontaneous mutations that activate the pathways affected by Gag could result in the low incidence of tumor development in mice infected with tumor-attenuated viruses.
Using genetic crosses between susceptible and resistant mice, we established that a single gene, mts, mapped to chromosome 14, determines the susceptibility of BALB/cJ mice to tumors induced by tumor-attenuated viruses.
Based on our preliminary data, we propose two models that could explain Gag/MTS cooperation in mammary tumor development. According to the first model (Fig. ), Gag binds directly to MTS, and this results in a signal transduction that cooperates with protooncogenes in mammary tumorigenesis. Due to allelic variance, BALB/cJ MTS can bind to either Mtv1
or MMTV(C3H) Gag, whereas C3H/HeN MTS can bind only to MMTV(C3H) Gag, and not to Mtv1
Gag (Fig. 7
FIG. 7. Model of how MTS may contribute to the induction of mammary tumors. MTS may contribute directly (A) or indirectly (B) to the induction of tumors by MMTV. MTS of the BALB/cJ origin may interact with both Gags in a similar fashion, whereas MTS of the C3H/HeN (more ...)
According to the second model (Fig. ), Gag binds to an unknown protein (factor X), and this binding activates MTS (most likely at the protein level). In this scenario, activation of C3H/HeN MTS requires a “stronger signal” (for example, high-affinity interaction) than does activation of BALB/cJ MTS. Engagement of factor X with MMTV(C3H) Gag, but not with Gag of Mtv1, fulfills this requirement in C3H/HeN mice. Because of the allelic difference, activation of BALB/cJ MTS does not require the same threshold of Gag/factor X interaction and thus could be induced in response to the Gag protein of Mtv1, as well as to the Gag protein of MMTV(C3H). However, we acknowledge that the situation may be more complex than the one described in Fig. . Since BALB/cJ mice are susceptible to tumors induced by all MMTV variants and C3H/HeN mice develop tumors only when infected with MMTV-encoding tumorigenic Gag [like MMTV(C3H)], one may infer that BALB/cJ MTS cooperates with both tumorigenic and tumor-attenuated Gags, whereas MTS of C3H/HeN origin is sufficiently activated only by a tumorigenic Gag (Fig. ). The induction of tumors by MMTV is not required for virus transmission and is a by-product of viral replication, indicating that the interplay between Gag and MTS may have some primary functions in the viral life cycle.
The primary function of Gag in all retroviruses is to produce all internal structural proteins. In addition, the MA protein of Gag demonstrates a nuclear export activity important for transporting unspliced viral RNA to the plasma membrane (13
). Finally, Gag proteins are the central players in the process of virion assembly (53
The complex and diverse activities of the Gag protein raise the possibility that Gag might interact with distinct cellular factors. The first evidence for interaction between Gag and host proteins was provided by studies of FV1, a dominant genetic host protein that limits the efficiency of integration of certain strains of murine leukemia viruses (46
). Viral sensitivity to this restriction is determined by sequences coding for CA (11
). A cellular factor that interacts with the Gag protein of murine leukemia virus has recently been cloned (2
). The gene contains a single intronless open reading frame with sequence similarity to the gag
gene of the ERV-L family of mouse and human endogenous retroviruses. Even though it remains unknown how FV1 blocks virus infection, one possibility is that the incoming virus is trapped by FV1 in a cytosol and is thus prevented from being transported into the nucleus. The human immunodeficiency virus CA protein was shown to interact with members of the cyclophilin family (30
). Mutations in the CA domain that abolish interaction with cyclophilin disrupt its incorporation into virions and preclude viral replication (29
). In mice, the activities of Gag are necessary and sufficient to induce immune system abnormalities in a syndrome designated mouse AIDS (1
). The Pr60gag
protein of the defective component of the mouse AIDS complex promotes the proliferation of infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, the SH3 domain of the ABL1 oncogene product was identified as interacting with the proline-rich p12 domain of Pr60gag
). Overexpression of Pr60gag
in these cells led to a detectable increase in the levels of ABL1 protein and to its translocation to the cell membrane. These results suggest that this viral Gag serves as a docking site for signaling molecules and that Abl1
may be involved in the proliferation of infected B cells. Gag proteins of Mason-Pfizer monkey virus, simian immunodeficiency virus, and human immunodeficiency virus type 1 have been found in association with a cellular motor protein, KIF4 (54
). It was suggested that KIF4 might be involved in the transport of Gag proteins in retrovirus-infected cells. Therefore, it is clear that diverse Gag-orchestrated retrovirus functions have the potential to interfere with numerous cellular pathways.
Retrovirus-induced tumors recapitulate pathways involved in the induction of tumors of other etiologies and thus provide a valuable model for the study of tumor induction in general. As already mentioned, cellular oncogenes, such as members of the Wnt
families, as well as ras
, and myc
, were originally identified in retrovirus-infected systems. Up-regulation of these oncogenes undoubtedly plays an important role in retrovirus-induced tumorigenesis. However, the induction of tumors, both spontaneous and of viral origin, always requires multiple steps. Our data suggest that the MMTV-encoded Gag protein plays a critical role in the induction of tumors by MMTV. The discovery that retrovirus-encoded proteins contribute to virus-induced tumors is also pertinent to other retroviruses, as recent findings involving Jaagsiekte sheep retrovirus implicate Env of Jaagsiekte sheep retrovirus in the induction of lung cancer in sheep (57
). Determination of the mechanisms by which retrovirus-encoded proteins contribute to tumor induction will help to identify cellular pathways that are involved in tumorigenesis in general.