Correction: Isa et al. HSV-1 ICP22 Is a Selective Viral Repressor of Cellular RNA Polymerase II-Mediated Transcription Elongation. Vaccines 2021, 9, 1054.

The authors would like to make the following corrections to this published paper [...].

Amber Suppression Technology for Mapping Site-specific Viral-host Protein Interactions in Mammalian Cells.

Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA. This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinity-purified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions. Graphic abstract: Schematic overview of the virus-host interaction assay based on an amber suppression approach. Mammalian cells grown in Bpa-supplemented medium are co-transfected with plasmids encoding viral sequences carrying a Flag tag, a (TAG) stop codon at the desired position, and an amber suppressor tRNA (tRNACUA)/aminoacyl tRNA synthetase (aaRS) orthogonal pair. Cells are then exposed to UV, to generate protein-protein crosslinks, followed by immunoprecipitation with anti-Flag magnetic beads. The affinity-purified crosslinks are probed by western blot using an anti-Flag antibody and the crosslinked host proteins are characterised by mass spectrometry.

HSV-1 ICP22 Is a Selective Viral Repressor of Cellular RNA Polymerase II-Mediated Transcription Elongation.

The Herpes Simplex Virus (HSV-1) immediate-early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint of a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells. Our findings indicate that ICP22 interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16, has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.