Transcriptional regulation of the murine cytomegalovirus m152 protein during acute infection

In eons of co-evolution, the species-adapted cytomegaloviruses (CMVs) have evolved multiple strategies dedicated to subvert the immune response. Most of the research done so far focused on the viral side, whereas much less is known on how the host counters viral immune evasion and to which extent viral and host proteins interact. The protein m152/gp40 was the first confirmed MHC-I-like immune evasion molecule encoded by mCMV and plays a central role in subverting antiviral immune responses. The transcription of m152 is controlled by an 'IFN regulatory factor element (IRFE)' located in the promoter region of the m152 gene. During acute infection, the IRFE is targeted by suppressive IFN response factors (IRFs) resulting in a delayed transcription of m152. This identified the m152 promotor region as a target for host countermeasures.

To identify transcription factors that regulate m152 gene expression, we have generated synthetic biotinylated DNA fragments, containing sections of the viral m152 promotor with potential transcription factor binding sites. The DNA-binding proteins were captured using an adapted pulldown protocol, which includes the best ratio of nuclear extract vs streptavidin-coated magnetic beads and target oligonucleotide, as well as optimized washing and eluting steps to reduce the background and increase the specificity of the pulldown. In brief, the biotinylated DNA was incubated with nuclear lysates of a murine fibroblast cell line (NIH3T3), followed by immobilization on streptavidin-coated magnetic beads. The DNA-binding proteins were eluted by digestion of the DNA using a nuclease. Afterwards, the proteins were digested using a Single-Pot, Solid-Phase-enhanced Sample Preparation (SP3) protocol and analyzed by LC-MS on a Bruker timsTOF Pro 2 mass spectrometer coupled to a nanoElute LC system in diaPASEF mode. Library-free search of raw data using UniProtKB/Swiss-Prot FASTA databases of Mus Musculus and Murid herpesvirus 1 (strain Smith) proteins and LFQ was performed in DIA-NN.

Current results from our DNA-protein pulldowns revealed several interferon regulatory factors (IRFs) that bind to the viral promotor sequence of m152 with IRFE region. These IRFs most likely regulate the transcription of the m152 during in the early stages of infection as a host countermeasure against viral infection. In addition, several more proteins were found to be binding to the oligonucleotide sequences, such as different subunits of nuclear factor NF-kappa-B and transcription factors, which may be forming complexes together with IRFs that regulate the transcription of m152.In conclusion, our current investigation has helped to shed light into the factors involved in transcriptional regulation of the viral m152 protein during the early stages of infection.