Mutational and Functional Analysis of the C-Terminal Region of the C3H Mouse Mammary Tumor Virus Superantigen

Abstract

The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vβ) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vβ chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+Vβ14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of Sag function may be compensated, at least partially, by loss of transcriptional suppression in certain tissues. Together these results confirm the importance of Sag for efficient milk-borne transmission and indicate that the entire C-terminal region is needed for complete Sag function.

Expression of Mouse Mammary Tumor Virus Superantigen mRNA in the Thymus Correlates with Kinetics of Self-Reactive T-Cell Loss

“…Clumps were removed on ice by settling for 5 min, and single cells were washed several times in FACS buffer before staining. Peripheral blood lymphocytes were obtained and purified as described previously (53,54). Cells (approximately 10 6 ) were incubated with antibody specific for V␤3, -5, -6, -7, -8, -9, -11, -12, or -14 labeled with fluorescein and CD4-or CD8-specific antibody labeled with phycoerythrin (PharMingen, San Diego, Calif.).…”

“…The mouse mammary tumor virus (MMTV) superantigen (Sag) protein is necessary for the efficient transmission of virus from infected mother's milk to the mammary glands of offspring (23,54). MMTV transmission requires infection of both B and T lymphocytes to facilitate transport of virus from the gut of newborns to mammary cells, and lymphocytes appear to be a reservoir of virus in the postnatal period prior to mammary gland development (7,23,26).…”

“…Viral infection of B cells in the gut-associated lymphoid tissue leads to the expression of Sag at the B-cell surface as a type II transmembrane glycoprotein (32,33). The C-terminal portion of Sag in conjunction with major histocompatibility complex (MHC) class II protein is recognized by entire classes of T cells that have specific ␤ chains as part of the T-cell receptor (TCR) (4,54,59). Experiments that switch the N-terminal and C-terminal regions of different Sags as well as site-directed Sag mutations suggest that the C-terminal 30 to 40 amino acids are critical for TCR interaction (38,54,59).…”

“…The C-terminal portion of Sag in conjunction with major histocompatibility complex (MHC) class II protein is recognized by entire classes of T cells that have specific ␤ chains as part of the T-cell receptor (TCR) (4,54,59). Experiments that switch the N-terminal and C-terminal regions of different Sags as well as site-directed Sag mutations suggest that the C-terminal 30 to 40 amino acids are critical for TCR interaction (38,54,59). Interaction of Sag with the TCR leads to a T-cell signaling pathway that results in the production of cytokines and/or T-cell proliferation (3,45,50).…”

Apobec-Mediated Retroviral HypermutationIn Vivois Dependent on Mouse Strain

“…Our previous results using splice donor site mutants of MMTV and TBLV in BALB/c mice indicated that MMTV-encoded Rem is involved in antagonizing proviral mutagenesis by Apobec family enzymes, including AID (18). Although Sag-mediated amplification of infected B and T lymphocytes is needed for efficient MMTV transmission and mammary cancer development (27, 30, 42), TBLV does not require Sag for the induction of T-cell lymphomas (38). Therefore, using Sag-independent TBLV, we tested the effect of Rem loss in multiple strains of genetically engineered mice that are available on the B6 background.…”