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. 2013 Mar;87(5):2639-47.
doi: 10.1128/JVI.03171-12. Epub 2012 Dec 19.

A short cis-acting motif in the M112-113 promoter region is essential for IE3 to activate M112-113 gene expression and is important for murine cytomegalovirus replication

Kareni J Perez  1 , Francisco Puerta Martínez, Ruth Cosme-Cruz, Neysa M Perez-Crespo, Qiyi Tang
Affiliations

A short cis-acting motif in the M112-113 promoter region is essential for IE3 to activate M112-113 gene expression and is important for murine cytomegalovirus replication

Kareni J Perez et al. J Virol. 2013 Mar.

Erratum in

Abstract

Immediate-early 3 (IE3) gene products are required to activate early (E)-stage gene expression of murine cytomegaloviruses (MCMV). The first early gene activated by IE3 is the M112-113 gene (also called E1), although a complete understanding of the activation mechanism is still lacking. In this paper, we identify a 10-bp cis-regulating motif upstream of the M112-113 TATA box as important for IE3 activation of M112-113 expression. Results from DNA affinity assays and chromatin immunoprecipitation assays show that the association of IE3 with the M112-113 gene promoter was eliminated by deletion of the 10-bp DNA sequence, now named IE3AM (for IE3 activating motif). In addition, IE3 interacts with TATA box binding protein (TBP), a core protein of TFIID (transcription initiation) complexes. Finally, we created an IE3AM-deleted MCMV (MCMVdIE3AM) using a bacterial artificial chromosome system. The mutant virus can still replicate in NIH 3T3 cells but at a significantly lower level. The defectiveness of the MCMVdIE3AM infection can be rescued in an M112-113-complemented cell line. Our results suggest that the interactions of IE3 with IE3AM and with TBP stabilize the TFIID complex at the M112-113 promoter such that M112-113 gene expression can be activated and/or enhanced.

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Figures

Fig 1
Fig 1
Mapping out the minimal size in M112-113 gene promoter that is essential for IE3 to activate M112-113 gene expression. (A) Diagram of the deletional mutation of the M112-113 gene; pBB5.5 contains the whole gene of M112-113, including promoter and open reading frame (ORF). Numbers on the left side of each construct stand for the length upstream from the initiation site of the M112-113 gene in the plasmid named on the left side. ORF112-113 stands for the open reading frame of M112-113. (B and C) Western blots to examine M112-113 gene production. NIH 3T3 cells were harvested 24 h after cotransfection of the plasmids indicated on the top of each picture. IE3 (expressed from pgfpie3) and GFP (expressed from pET) were detected by anti-GFP antibody, while M112-113 was examined by anti-M112-113 antibody. Tubulin was used as a sample loading control. (D) Real-time RT-PCR to examine M112-113 gene expression. Total RNAs were isolated from NIH 3T3 cells after 24 h of cotransfection of M112-113-expressing plasmids (as indicated) with pgfpie3 or pET. The CT values first were normalized to that of beta-actin, and then comparisons were made between pgfpie3 and pET groups to calculate the ΔΔCT. Relative quantity was shown as R = 2−ΔΔCT.
Fig 2
Fig 2
Identification of a DNA motif that is required for IE3 to activate M112-113 gene expression. (A) Partial DNA sequence before the TATA box (boxed) in the M112-113-expressing vectors. Black lines represent the DNA sequences deleted. (B) Western blot assay to examine the activation of the M112-113 gene by IE3. The experimental methods are the same as those for Fig. 1B and C. (C) Real-time RT-PCR to examine the activation of the M112-113 gene by IE3. The experimental methods are the same as those for Fig. 1D.
Fig 3
Fig 3
Ten-bp DNA motif mediates the interaction of IE3 with TBP and stabilizes TFIID complexes. (A and B) DNA ChIP assay to examine IE3-DNA interaction. (A) M112-113 gene structure is shown at the top; input DNA was amplified to show the total DNA in the samples, and IgG-incubated samples were used as a negative control. In the bottom panel, ChIP DNA using anti-GFP shows that IE3-bound DNA is the strongest in the region (F) where the 10-bp motif resides. (B) Real-time PCR was performed to show the percentage anti-GFP ChIP DNA compared to the amount of the input. Results are consistent with those of regular PCR shown in A. (C) DNA affinity assay to ascertain direct interaction of IE3 with the M112-113 promoter with or without the 10-bp DNA. (Left side) DNA affinity assay with the whole nuclear extract (NE). Mock indicates the NE prepared from uninfected NIH 3T3 cells. Input was NE made from MCMV-infected (MCMV Inf.) NIH 3T3 cells. (Right side) DNA affinity assay with purified proteins (Puri. Pr.) of TBP (upper) and GFPIE3 (lower). (D) Coimmunoprecipitation assay to determine the interaction of TBP with IE3. Nuclear extracts were prepared from NIH 3T3 cells infected with MCMVE5gfp at a multiplicity of infection of 1 and incubated with antibodies as indicated at the top of the panel. The pulled-down proteins were detected by Western blotting using antibodies shown on the right side. For the detection of protein in the immunoprecipitation, we used secondary antibodies from TrueBlot ULTRA (18-8817 for mouse or 18-8816 for rabbit; eBioscience) to avoid the heavy chain.
Fig 4
Fig 4
Comparative protein production of MCMVE5gfp, MCMVE5gfpdIE3AM, and MCMVE5gfpdIE3AMRQ. (Upper) The viral DNAs of MCMVE5gfpdIE3AM and MCMVE5gfpdIE3AMRQ were isolated and sequenced for the whole M112-113 region, confirming that IE3AM is removed from MCMVE5gfpdIE3AM and restored in MCMVE5gfpdIE3AMRQ, and that the DNA sequences of the whole region are correct. (Lower) Three viruses were used to infect NIH 3T3 cells, and the cells were harvested at the indicated time points. Viral proteins were detected by Western blotting using antibodies against the viral proteins shown on the right side (IE1, GFPIE3, m25, M44, and M112-113). Tubulin was used for controlling sample loading.
Fig 5
Fig 5
Viral PFU assay. (A) Viral replication ability in NIH 3T3 cells. MCMVE5gfp, MCMVE5gfpdIE3AM, or MCMVE5gfpdIE3AMRQ was used to infect NIH 3T3 cells, and the cells were collected on the days postinfection indicated. After three cycles of freezing and thawing, the cell-medium mixture was centrifuged and the supernatant was used for PFU assays. Each experiment was performed independently three times, and the mean number of PFU was obtained by averaging the numbers from different dilutions. (B) The same as A, but infection was carried out in the NIH 3T3e1 cell line.
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