An Unbiased Approach to Mapping the Signaling Network of the Pseudorabies Virus US3 Protein

Abstract

The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle.

Alphaherpesvirus US3 protein-mediated inhibition of the m6A mRNA methyltransferase complex

“…Expression of the US3 kinase, but not the UL13 kinase, triggered phosphorylation of both METTL3 and METTL14 ( Figure 2F ). Note that the poor expression of the kinase-inactive version of the US3 kinase, in line with earlier reports ( Jansens et al, 2020 ), prevented conclusions regarding the kinase dependency of this phosphorylation.…”

The Deletion of US3 Gene of Pseudorabies Virus (PRV) ΔgE/TK Strain Induces Increased Immunogenicity in Mice

“…Therefore, the current commercial live-attenuated PRV vaccines are produced on the basis of the deletion of gE and TK [ 6 , 7 , 8 ]. US3 is also a virulent gene of PRV, and protein encoded by US3 could help PRV to escape immune surveillance of the host, by inhibiting type I IFN expression, interfering with MHC I-mediated antigen presentation and preventing infected cells from apoptosis [ 9 , 10 , 11 ]. Therefore, PRV ΔgE/TK/US3 strain could potentially be developed into an effective commercial live-attenuated vaccine.…”

Pseudorabies Virus Infections

“…Studies on PrV repeatedly pioneered research on basic principles of herpesvirus replication. Data on the complex transcriptome [9], the functional role of N-linked glycans of glycoprotein gB [10] and an unbiased approach to uncover the still unknown signaling network of the pUS3 protein kinase [11] will stimulate further research on the molecular specifics of PrV and other (alpha-)herpesviruses.…”