C3H Mouse Mammary Tumor Virus Superantigen Function Requires a Splice Donor Site in the Envelope Gene

Mouse mammary tumor virus (MMTV) is transmitted from the milk of infected mothers to the gut of susceptible offspring (33). MMTV infects B cells in the guts of newborn mice, and these cells express the virally encoded superantigen (Sag) at the plasma membrane in association with the major histocompatibility complex (MHC) class II protein (1, 28). Sag is a type II transmembrane protein that is required for efficient transmission of milk-borne MMTV from the gut to the mammary gland (12, 17, 23). The Sag-MHC complex is recognized by entire classes of T cells bearing particular T-cell receptor (TCR) β chains (18, 28). Recognition of Sag by specific T cells leads to cell proliferation and/or release of cytokines, and the released cytokines recruit additional B and T lymphocytes that are infected by MMTV (27). B-cell-deficient mice or mice lacking Sag-reactive T cells cannot be efficiently infected by MMTV (4, 12). Ultimately, viral infection of the mammary gland is necessary to allow MMTV release into the milk. Mice that lack B cells or Sag-specific T cells also are defective in the spread of MMTV within the mammary gland (14). Thus, Sag is required for generation of a reservoir of virally infected B and T lymphocytes that are involved in MMTV transmission and viral spread to the mammary tissues. The regulation of MMTV sag expression is controversial. Early studies indicated that Sag is translated from a singly spliced mRNA that initiates at the U3/R border of the viral long terminal repeat (LTR) from the predominant U3 promoter (21, 41). This U3 promoter also drives expression of the viral structural genes, gag, pol, and env (for a review, see reference 10) (Fig. ​(Fig.1).1). Cloning and sequencing revealed that this sag mRNA uses a single splice donor site in the leader region that also is used for the generation of spliced envelope mRNA (32). The splice acceptor site for this sag mRNA is located in the envelope region just upstream of the 3′ LTR (21). Multiple start codons are located near the 5′ end of this mRNA, and mutagenesis experiments have suggested that the first or second codons can suffice for functional Sag production in cell culture (8). Subsequently, at least three other potential sag mRNAs have been described (Fig. ​(Fig.1).1). One of these mRNAs uses the same splice donor and acceptor sites as those described earlier, except that this transcript initiates approximately 500 bp upstream of the standard viral RNAs (16). Another transcript initially was identified as a phorbol ester-inducible, cyclosporine-suppressible RNA in a T-cell lymphoma (11, 31, 38). This sag-specific RNA is initiated from an intragenic promoter within the envelope-coding region; the transcript uses a unique splice donor site within the envelope region and the same splice acceptor site as the other two sag RNAs. Most recently, a different sag promoter has been described within the envelope region (2). This promoter was shown to be active in transient transfection experiments with reporter gene constructs. The resulting sag mRNA appears to be unspliced and initiated within 100 bp of the 3′ LTR (2). FIG. 1 Diagram of the MMTV sag-specific mRNAs and the location of the splice donor mutation. (A) Schematic representation of the MMTV genome and reported MMTV sag mRNAs. The boxes on the MMTV proviral genome show the positions of the indicated open reading frames ... Using PCR assays, our experiments have shown that all of the spliced sag transcripts are detectable in lymphocytes of BALB/c mice that contain three endogenous MMTVs, Mtv-6, Mtv-8, and Mtv-9 (9, 24). However, only the spliced sag transcript from the envelope promoter was detectable in cells infected in vitro or in vivo with milk-borne C3H MMTV. Similarly, deletion mutants of a C3H MMTV-derived infectious molecular clone (39) suggested that an intragenic envelope promoter and an enhancer in the pol gene were responsible for sag expression (34, 35). To test whether the spliced mRNA from the env promoter is required for C3H MMTV sag expression, we used the C3H-derived infectious molecular clone to construct a mutant with alterations in the splice donor site within the envelope region that is unique to this transcript. Stable transfections of this mutant or a control frameshift mutant with an alteration within the sag coding region that has been shown to abolish Sag function (13) produced virus-expressing cell lines. Both the splice donor and frameshift mutants induced some mammary tumors with long latency and sporadic T-cell deletion after direct injection of transfected cells. However, neither the frameshift nor the splice donor mutant was transmissible to susceptible third-litter progeny through the milk-borne route. These experiments show that the spliced mRNA from the envelope region is necessary and sufficient for efficient C3H MMTV milk-borne transmission.