ABSTRACT
The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Cell Nucleus/metabolism , Genome, Viral , Herpesvirus 1, Suid/physiology , Viral Proteins/metabolism , Actin Cytoskeleton/ultrastructure , Animals , Cells, Cultured , Cytochalasin D/pharmacology , Genome, Viral/drug effects , Herpesvirus 1, Suid/drug effects , Herpesvirus 1, Suid/genetics , Male , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pseudorabies/virology , Rabbits , Real-Time Polymerase Chain Reaction , Swine , Testis/cytology , Viral Proteins/genetics , Virion/genetics , Virus InternalizationABSTRACT
The conserved alphaherpesvirus serine/threonine kinase US3 causes dramatic changes in the actin cytoskeleton, consisting of actin stress fibre breakdown and protrusion formation, associated with increased virus spread. Here, we showed that US3 expression led to RhoA phosphorylation at serine 188 (S188), one of the hallmarks of suppressed RhoA signalling, and that expression of a non-phosphorylatable RhoA variant interfered with the ability of US3 to induce actin rearrangements. Furthermore, inhibition of cellular protein kinase A (PKA) eliminated the ability of US3 to induce S188 RhoA phosphorylation, pointing to a role for PKA in US3-induced RhoA phosphorylation. Hence, the US3 kinase leads to PKA-dependent S188 RhoA phosphorylation, which contributes to US3-mediated actin rearrangements. Our data suggest that US3 efficiently usurps the antagonistic RhoA and Cdc42/Rac1/p21-activated kinase signalling branches to rearrange the actin cytoskeleton.
Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Herpesvirus 1, Suid/enzymology , Protein Serine-Threonine Kinases/metabolism , Pseudorabies/enzymology , Swine Diseases/enzymology , Viral Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Herpesvirus 1, Suid/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Pseudorabies/metabolism , Pseudorabies/virology , Swine , Swine Diseases/metabolism , Swine Diseases/virology , Viral Proteins/genetics , rhoA GTP-Binding Protein/chemistry , rhoA GTP-Binding Protein/geneticsABSTRACT
Rho GTPases are key regulators of actin and microtubule dynamics and organization. Increasing evidence shows that many viruses have evolved diverse interactions with Rho GTPase signaling and manipulate them for their own benefit. In this review, we discuss how Rho GTPase signaling interferes with many steps in the viral replication cycle, especially entry, replication, and spread. Seen the diversity between viruses, it is not surprising that there is considerable variability in viral interactions with Rho GTPase signaling. However, several largely common effects on Rho GTPases and actin architecture and microtubule dynamics have been reported. For some of these processes, the molecular signaling and biological consequences are well documented while for others we just begin to understand them. A better knowledge and identification of common threads in the different viral interactions with Rho GTPase signaling and their ultimate consequences for virus and host may pave the way toward the development of new antiviral drugs that may target different viruses.
Subject(s)
rho GTP-Binding Proteins/metabolism , Actins/metabolism , Clathrin/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Endocytosis , Humans , Microtubules/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Viruses/metabolism , Viruses/pathogenicityABSTRACT
The conserved alphaherpesviral serine/threonine kinase US3 causes dramatic actin rearrangements, associated with increased viral spread. Here, we show that US3 of pseudorabies virus (PRV) leads to activation (dephosphorylation) of the central actin regulator cofilin. A mutation that impairs US3 kinase activity and the group I p21-activated kinase inhibitor IPA-3 inhibited US3-mediated cofilin activation. Additionally, expression of phosphomimetic S3D cofilin significantly suppressed the ability of US3 to cause cell projections and cell rounding. In conclusion, the US3 kinase of PRV leads to activation (dephosphorylation) of cofilin, and cofilin contributes to US3-mediated actin rearrangements.
Subject(s)
Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Herpesvirus 1, Suid/enzymology , Protein Serine-Threonine Kinases/metabolism , Virus Internalization , Blotting, Western , Cell Shape/physiology , Enzyme Inhibitors/metabolism , Microscopy, Fluorescence , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , p21-Activated Kinases/antagonists & inhibitorsABSTRACT
Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.
