Mouse mammary tumor virus superantigen expression in B cells is regulated by a central enhancer within the pol gene

Abstract

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. The mechanism of superantigen expression from the viral sag gene in B cells is largely unknown, due to problems with detection and quantification of these low-abundance proteins. We have established a sensitive superantigen-luciferase reporter assay to study the expression and regulation of the MMTV sag gene in B-cell lymphomas. The regulatory elements for retroviral gene expression are generally located in the 5' long terminal repeat (LTR) of the provirus. However, we found that neither promoters nor enhancers in the MMTV 5' LTR play a significant role in superantigen expression in these cells. Instead, the essential regulatory regions are located in the pol and env genes of MMTV. We report here that maximal sag expression in B-cell lines depends on an enhancer within the viral pol gene which can be localized to a minimal 183-bp region. Regulation of sag gene expression differs between B-cell lymphomas and pro-B cells, where an enhancer within the viral LTRs is involved. Thus, MMTV sag expression during B-cell development is achieved through the use of two separate enhancer elements.

FIG. 1

Schematic representation of the MMTV provirus, Sag-reporter proviruses, and regulatory elements for MMTV sag gene expression in B cells. The positions of viral genes (gag, pro, pol, env, and sag), promoters (P₆₉₈, P₁₁₉₆, P₇₂₄₆, and P₈₄₉₈), newly characterized pol gene enhancer (E), and functional regions within the LTRs (U3, R, and U5) are indicated. An additional stimulatory element (hatched box) is located between E and P₇₂₄₆. The Sag-reporter proviruses M^sag17-luc (sag17-luc) and M^sag1-luc (sag1-luc) differ from the MMTV provirus in the 3′ LTRs only. The recombinant 3′ LTRs are depicted below the original LTR. The position of the reporter gene firefly luciferase (luc) in the recombinant constructs is shown. Regulatory elements with demonstrated relevance for sag gene expression are represented as open and hatched boxes.

FIG. 2

MMTV sag-reporter activity in cell lines representing pro-B, pre-B, and mature stages of B-cell development. MMTV sag-reporter proviruses M^sag17-luc (sag17-luc) and M^sag1-luc (sag1-luc), and positive control plasmid CMV-luc were transfected into the pro-B-cell line Ba/F3, the pre-B-cell lines 300-18 and 2M3/M, the B-lymphoma lines A20, CH28, and M12, and the B-cell hybridoma LBB.11. We used 2 pmol of each test plasmid, except for CMV-luc, for which we used 0.5 pmol of plasmid. The full-length MMTV reporter constructs are denoted by plus signs, whereas the negative control constructs lacking the 5′ LTR and most of the internal region are represented by minus signs. Cells were harvested after 24 h and lysed. The relative luciferase activity (RLU) and protein concentration were determined with this extract. The numbers represent the arithmetic mean ± standard deviation (SD) of at least three separate experiments with two different DNA preparations.

FIG. 3

Functional analysis of MMTV proviral regions that are involved in sag gene expression by 5′ truncation analysis of MMTV sag-reporter provirus Msag17-luc. The sag-luciferase reporter provirus M^sag17-luc, a series of 5′ truncation mutants (1459, 1637, 2412, 3246, 4251, 5055, 5803, 7478, and 8532), or positive control plasmid CMV-luc were transfected into Ba/F3 pro-B cells, A20 B-cell lymphoma, or LBB.11 B-cell hybridoma. We used 2 pmol of each test plasmid, except for CMV-luc, for which we used 0.5 pmol of plasmid. Cells were harvested after 24 h, and the lysate was tested for luciferase activity and protein concentration. The numbers represent the arithmetic mean ± SD of at least three separate experiments with two different DNA preparations. The MMTV proviral construct is depicted below the graph. The positions of the viral genes (gag, pol, env, and sag) and reported promoters (P) are indicated. The relative result for each truncation mutant is represented at the position of the truncation endpoint within the MMTV provirus.

FIG. 4

MMTV Sag expression in B cells is independent of the sag gene intragenic enhancer. Reporter plasmids pM^sag17-luc(E_proB^+/+) (open circles) and pM^sag17-luc(E_proB^+/−) (open squares), with their respective truncation mutants Δ5055, Δ5803, Δ7478, and Δ8532, and luciferase control plasmid CMV-luc (solid squares) were transfected into either Ba/F3 pro-B cells (top) or B-cell hybridoma LBB.11 (bottom). The mock transfection is represented by solid circles. We used 2 pmol of each test plasmid, except for CMV-luc, for which we used 0.5 pmol of plasmid. Cells were harvested after 24 h, and luciferase activity was determined. Results are expressed as relative luciferase units (RLU) per microgram of protein and represent the arithmetic mean ± SD of at least three separate experiments with two different DNA preparations. The viral constructs with nucleotide positions are depicted below the graphs. The approximate positions of coding regions (gag, pol, and env), promoters (P), and reporter gene luciferase (luc) are shown. The specific constructs transfected are identified above the graphs.

FIG. 5

The MMTV pol gene contains an enhancer element for gene expression in B cells. (A) Stimulation of SV40 promoter-directed luciferase expression by an MMTV pol gene fragment (nt 5055 to 6255). (B) Schematic representation of enhancer test constructs pGL2-pol. Control plasmid pGL2-promoter (VE) contains an enhancerless SV40 promoter upstream of the firefly luciferase reporter gene. The MMTV pol gene fragment (nt 5055 to 6255) was introduced in a position either 5′ or 3′ from the SV40 promoter-luciferase reporter gene of enhancer test plasmid pGL2-promoter, in either the sense (+) or antisense (−) orientation, to generate pGL2-pol 5+, 5−, 3+, and 3−, respectively. These plasmids, pGL2.promoter (VE) and CMV.luc (PC), were transfected into B-cell lines LBB.11, M12, and A20. We used 2 pmol of each test plasmid, except for CMV-luc, for which we used 0.5 pmol of plasmid. The cells were harvested after 24 h (A20 and LBB.11) or 48 h (M12), and the luciferase activity and protein concentrations in the cell lysate were determined. Results are expressed as relative luciferase units (RLU) per microgram of protein and represent the arithmetic mean ± SD of at least three separate experiments with two different DNA preparations. As previously established (50a), introduction of an irrelevant piece of DNA into pGL2-promoter does not result in increased luciferase expression.

FIG. 6

Localization of the functional B-cell enhancer region within the MMTV pol gene. (A) Enhancer activity of MMTV pol gene regions. (B) Nucleotide sequence of the MMTV pol gene enhancer region. The MMTV pol region (nt 5055 to 6255) in pGL2-pol 3+ was successively truncated from the 5′ terminus (solid arrowhead) and from the 3′ terminus (open arrowhead). The truncation mutants were transfected into LBB.11 B cells. After 24 h, the cells were harvested and lysed and the luciferase activity and protein concentrations determined. The numbers indicate the position of the test fragment within the MMTV provirus. Specific truncation mutants are identified below the graph for the 5′ truncation series (solid circles) and above the graph for the 3′ truncations (open circles). The truncation mutant 6252 represents the vector control and is identical to the plasmid pGL2-Promoter (Promega). The bars below the graph represent the pol gene fragment analyzed, with the shaded portions indicating the parts required for enhancer activity in the respective 5′ and 3′ truncations and the hatched portions specifying the dispensable parts. Approximate positions of the env gene and splice acceptor (SA) site are indicated.

FIG. 7

Effect of the MMTV pol gene enhancer and env gene promoter P₇₂₄₆ on sag gene expression in a complete provirus. An intact MMTV provirus (MMTV) and deletion mutants lacking the pol gene enhancer (ΔE), env gene promoter P₇₂₄₆ (ΔP), both elements (ΔE+P), or the pol gene enhancer, P₇₂₄₆ and the intervening region (ΔE to P), the negative control pM^sag17-lucΔ8532 (Δ8532), or control plasmid CMV-luc were transfected into the B-cell lines A20, LBB.11, and M12. We used 2 pmol of each test plasmid, except for CMV-luc, for which we used 0.5 pmol of plasmid. After 24 h, the cells were harvested and lysed, and luciferase activity and protein concentrations were determined. Results are expressed as relative luciferase units per microgram of protein and represent the arithmetic mean ± SD of at least three separate experiments with two different DNA preparations.