The US3 protein is a viral kinase that is conserved among the Alphaherpesvirinae. Here, we show that US3 of the swine alphaherpesvirus pseudorabies virus causes dramatic alterations in the cytoskeleton, resulting in the formation of long actin-and microtubule-containing cell projections in infected and transfected cells. Analysis with a GFP-labeled virus showed that multiple virus particles move inside the projections toward the tip. GFP-labeled virus could also be found in the cytoplasm of neighboring cells that were in contact with the projections. In addition, projection formation could be inhibited by using the actin-stabilizing drug jasplakinolide and could be induced by using the Rho kinase inhibitor Y27632. Analyzing the effect of these drugs on intercellular virus spread indicated that the observed US3-induced alterations in the host cytoskeleton are associated with enhanced intercellular virus spread, thereby suggesting a previously undescribed aspect of alphaherpesvirus spread.cytoskeleton ͉ herpes ͉ projections

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXX⌽ motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.Pseudorabies virus (PRV) is a swine alphaherpesvirus which, like most alphaherpesviruses, has evolved in several ways to subvert the immune system of its host. One noteworthy example of PRV immune modulation is the ability to replicate in the respiratory tracts of vaccinated animals. A viremia often results, giving rise to striking PRV symptoms, including abortion (24, 39). This viremia in vaccinated pigs requires cell-to-cell spread of PRV in tissue and transport of virus via infected monocytes in the blood (23,24,25,39).Mechanisms used by PRV, as well as by the prototypical alphaherpesvirus herpes simplex virus (HSV), to avoid recognition and destruction by the immune system include strategies to downregulate major histocompatibility complex class I-dependent antigen presentation in infected cells (3, 33; for a review, see reference 40), direct cell-to-cell spread of the virus, binding of complement factors via viral glycoprotein C (gC), Fc receptor activity of viral glycoprotein complex gE-gI, and, for PRV, the recently described antibody-induced internalization of viral cell surface proteins in PRV-infected blood monocytes, the natural carrier cells of the virus in vaccinated animals (9,10,14,15,17,18).For PRV, two of these mechanisms are mediated by viral glycoprotein gB: (i) the antibody-induced internalization of viral cell surface glycoproteins (a rapid and massive internalization of the majority of plasma membrane-anchored viral proteins upon aggregation of these proteins caused by the addition of PRV-specific antibodies, a process that likely results in inefficient antibody-dependent lysis of PRV-infected monocytes) and (ii) the direct cell-to-cell spread of the virus (10,29,31,38).PRV gB is a type I membrane glycoprotein of 913 amino acids (aa), consisting of an extracellular domain, a transmembrane region, and a 93-aa cytoplasmic C-terminal tail. At least three putative endocytosis motifs located within the cytoplasmic tail of gB are conserved throughout the alphaherpesvirus family. Two are tyrosine-based YXX⌽ sequences (where Y stands for tyrosine, X stands for any amino acid, and ⌽ represents a bulky, hydrophobic group) and one is a dileucine (LL) motif. YXX⌽ and LL motifs in the c...

Disruption of specific components of the host cytoskeleton has been reported for several viruses and is thought to be beneficial for viral replication and spread. Our previous work demonstrated that infection of swine kidney (SK-6) cells with pseudorabies virus (PRV), a swine alphaherpesvirus, induced actin stress fiber breakdown. In the present study, using several PRV deletion mutants, we found that the US3 serine/threonine (S/T) protein kinase is involved in breakdown of actin stress fibers in different PRV-infected cell lines. Further, by transfection assays, we showed that PRV US3 itself, in the absence of other viral proteins, is able to trigger actin stress fiber breakdown when it is localized in sufficient amounts in the nucleus.

Core facilities have become an important resource in biomedical research, providing scientists with access to sophisticated instrumentation and expertise. To enable scientists to perform ever more complex and difficult experiments, core facilities not only need to constantly upgrade technology and expertise, but also cooperate and pool their assets.