Mouse cytomegalovirus early M112/113 proteins control the repressive effect of IE3 on the major immediate-early promoter

Abstract

The mouse cytomegalovirus major immediate-early (IE) transcript is differentially spliced to produce two IE proteins: IE1, which functions partly to maintain its own promoter, the major IE promoter (MIEP), free from repression, and IE3, which functions partly as a repressor of MIEP. Paradoxically, the site where transcription of the viral genome occurs is also the site where the greatest amounts of IE3 accumulate. This raises the question of how the repression capabilities of IE3 are controlled so soon after infection. We detected IE3, an activator of early proteins, contemporaneously with gene products of the early M112/113 locus. Both IE3 and the early M112/113 gene products colocalize and coimmunoprecipitate. Protein interaction most likely occurs between IE3 and the 87-kDa splice form of M112/113, because only the 87-kDa component coimmunoprecipitated with IE3. The complex also includes PML. Transiently expressed M112/113 can form large domains alone, even in the absence of full viral genomes or PML. Coexpression of M112/113 products and IE3 results in segregation of IE3 into newly formed M112/113-based domains. Importantly, coexpression eliminates the IE3-based repressive effect on MIEP, as determined by MIEP-driven reporter assays. The consequence of segregating IE3 into the M112/113-containing prereplication domains appears to make IE3 unavailable for binding and repressing MIEP during the earliest stages of infection. These findings establish a new feedback mechanism between IE and early proteins, a new mechanism of promoter control via segregation of the repressor, and a new function for proteins from the M112/113 locus.

FIG. 1.

Segregation of IE3 and M112/113 during infection and transient expression. (A) MCMV-infected 3T3 cells at 2 h p.i. In situ hybridization with IE DNA as a probe. IE transcripts localize adjacent to anti-PML-immunostained ND10. (B) The experiment as described in panel A, but cells are stained for ND10 with anti-PML and anti-M112/113 antibodies. M112/113 appears adjacent to ND10. (C) M112/113-transfected 3T3 cells labeled for ND10 and M112/113. M112/113 localizes adjacent to PML at early times after transfection (12 h). (D) IE3-GFP appears adjacent to ND10 at early times after transfection (12 h). (E) IE3-GFP and 112/113 colocalize at early times after transfection (12 h). (F) MCMV-infected 3T3 cells at 24 h p.i. were stained for M112/113 (red) and PML (blue), followed by fluorescence in situ hybridization (green). Replicated viral DNA is located in M112/113- and PML-containing replication compartments. (G to I) 3T3 cells transfected with plasmids expressing M112/113 are shown stained for PML in panel H and for M112/113 in panel I. M112/113 forms large domains with PML present at its center. (J) 3T3 cells transfected with plasmids expressing M112/113 and IE3-GFP. M112/113 and IE1-GFP colocalize and form large domains. (K) The experiment is the same as described for panel J. M112/113 is shown alone. (L) 3T3 cells infected with MCMV at 24 h p.i. and immunostained for M112/113 and by in situ hybridization for total viral DNA. The start of replication is evident in the lower left cell. Very limited replication is apparent in the lower right cell, whereas no signs of replication are apparent in the top cell. (M) Higher magnification of the top cell of panel L.

FIG. 2.

Coimmunoprecipitation of proteins present in the replication compartment. 3T3 cells were transfected with M112/113 or M112/113 and IE3-GFP and then lysed, and their extracts were immunoprecipitated with either anti-M112/113 antibodies or anti-GFP antibodies. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated proteins, Western blots were prepared and probed with the antibodies indicated on the left after successive stripping of the same transfer membrane. Lane 1, 5% input; lane 2, extract immunoprecipitated with nonspecific serum control; lane 3, extracts immunoprecipitated with anti-M112/113 antibodies; lane 4, extracts immunoprecipitated with anti-GFP antibodies (IE3). IP, immunoprecipitate.

FIG. 3.

Western blotting assessment of the effect of M112/113 on IE3 expression. The amount of IE1 (lanes 2 and 3) and IE3 (lanes 4 and 5) transcribed under the direction of MIEP was assessed in cells not expressing M112/113 (lanes 1, 2, and 4) and cells expressing M112/113 (lanes 3 and 5). The GFP tags of IE1-GFP and IE3-GFP were immunostained with anti-GFP antibodies. The stripped transfer membrane was immunostained with rabbit anti-112/113 antibody to detect the expression of M112/113 and antitubulin antibody to detect the expression of the housekeeping gene tubulin. Note the larger amount of IE3 in lane 5 compared to that in lane 4. The nonspecific band at ∼36-kDa is due to the rabbit antibodies.

FIG. 4.

Modification of IE3-mediated repression of MIEP by M112/113 proteins. (A) In bars 1 to 3, the presence or absence of M112/113 in 3T3 cells cotransfected with pmMIEP-luc and pBB5.5(e1), which expresses M112/113 products, did not affect luciferase activity (bars 1 and 2). Cells transfected with the pET vector (bar 4) producing GFP showed no difference in luciferase activity compare to cells transfected with empty vector controls. When IE3 was expressed in cells transfected with pmMIEP-luc, luciferase expression decreased (compare bars 4 and 5). When 3T3 cells were cotransfected with pBB5.5, which expresses M112/113 proteins, and with pmMIEPluc, luciferase activity was similar to the activity level of controls (bars 4 and 6). Cotransfection of the luciferase reporter and pGFF-tagged IE1 enhanced luciferase activity above the activity observed in controls (bars 4 and 7). pUC18 was used to adjust the amount of DNA for transfection. (B) Western blot of the M112/113 proteins expressed in 3T3 cells harboring different combinations of vectors, as indicated in panel A (lanes of the blot correspond to bars 3 to 7 of panel A). The published molecular sizes of M112/113 proteins are indicated to the left of the blot. (C) Western blot of the blot shown in panel B. The stripped membrane was reprobed with anti-GFP antibodies to detect GFP-labeled IE3 (lane 5 and 6) or GFP-labeled IE1 (lane 7). Note the larger amount of IE3 in lane 6 compared to that in lane 5.