Steroid Hormone Receptors

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doi: 10.1128/JVI.02983-13. role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression. IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases. failed to rescue the gene-deficient recombinant virus (20, 30). The imPS system is useful and convenient for overcoming such issues. Briefly, a protein destabilization domain and one half of the split intein is fused to the N or C terminus of the gene of interest. The protein products of the fusion gene will be quickly degraded and lose function. However, expressing the other half of split intein in reconstitutes the enzymatically active intein, thereby mediating cleavage of the protein destabilization domain, rescuing the gene product and virus replication. In this work, we inserted MM-102 TFA the split intein into the middle of pM49 coding sequence because its N-terminal coding sequence overlaps with M50, whereas 3FLAG tag C-terminal insertion slightly affects functional output. As shown in Fig. 2A, the coding sequence of gp41-1 N-terminal split intein (IntN; gray box) and a stop codon (TAG; black box) were inserted at 793 nucleotides downstream of the pM49 start codon. The MM-102 TFA recombinant pM49-deficient virus SMdd49 expressed a truncated pM49 containing the N-terminal 264 amino acids of the full-length protein, with IntN fused to its C terminus (pM49-N-IntN).To reconstitute the full-length pM49, we MM-102 TFA constructed a lentiviral vector that expressed the gp41-1 C-terminal split intein (IntC) fused with the C-terminal half of the pM49 coding sequence (IntC-pM49-C). MEF10.1 cells were transduced with this lentiviral vector to generate complementary cells expressing the IntC-pM49-C fusion protein (Fig. 2B). When the pM49-deficient bacterial artificial chromosome (BAC) pSMdd49 was transfected into these complementary cells, pM49-N-IntN expressed from the BAC genome and IntC-pM49-C expressed from the lentiviral vector interacted to reconstitute the full-length pM49 via protein splicing (Fig. 2B and ?andC).C). As shown in Fig. 2C, transfection of pSMdd49 into MEF10.1control cells led to green fluorescent protein (GFP) expression in scattered individual cells at 5?days postinfection. In contrast, transfection of pSMdd49 into IntC-pM49-C-expressing complementary cells led to the spread of GFP expression in all cells, similar to that of wild-type BAC pSMgfp transfection into Tnf control or complementary cells (Fig. 2C). As expected, growth curve analyses showed that no SMdd49 virus was recovered from MEF10.1 control cells, while SMdd49 virus collected from IntC-pM49-C-expressing complementary cells grew to wild-type virus levels (Fig. 2D). These data suggest that full-length pM49 was essential for MCMV replication. Open in a separate window FIG 2 pM49 is essential for MCMV replication. (A) Schematic depicting the structure of pSMdd49 BAC used this study. pM49 coding sequence was inserted with a gp41-1 split intein N (IntN, gray box) coding sequence and a stop codon (TAG, black box) at 793?nt downstream of the start codon, resulting in an interrupted protein translation and the production of pM49-N-IntN. (B) Complementary model of the pM49-deficient virus (SMdd49) using the intein-mediated modulation of protein stability (imPS) system. Without Intc-pM49-C, cells infected with SMdd49 only expressed the fusion protein of pM49-N-IntN. However, intein splicing that occurred in complementary cells produced full-length pM49 to support viral growth. (C and D) MEF10.1Ctrl and MEF10.1 Intc-pM49-C cells were infected with SMgfp or SMdd49 at an MOI.