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.

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The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.Pseudorabies virus (PRV), a swine alphaherpesvirus closely related to the human pathogens herpes simplex virus (HSV) and varicella-zoster virus (VZV), is the causative agent of Aujeszky's disease (4, 24). Its genome encodes at least 11 glycoproteins, which have homologs in other herpesviruses (24). In PRV-infected cells, newly synthesized glycoproteins travel from the endoplasmic reticulum via the Golgi to the plasma membrane (25). These glycoproteins play important roles in the viral life cycle, as well as in the pathogenesis of PRV infections (9, 29).Interestingly, several alphaherpesvirus-encoded cell surfaceassociated envelope glycoproteins have been reported to be internalized, either spontaneously or upon binding of antigenspecific antibodies (12,13,17,32,35,36,44). The biological function of spontaneous internalization in the virus life cycle is not yet fully understood, although some hypothetical roles have been proposed (reviewed in reference 9), such as the possible involvement of internalization in delivering the viral cell surface proteins to a specific compartment, where viral envelopment takes place; in redirecting viral proteins to specific membrane surfaces (such as the apical, lateral, or basal surfaces of polarized cells); or in immune evasion. Antibodydependent internalization of viral cell surface proteins may also be implicated in immune evasion, since it has been shown to decrease the efficiency of antibody-dependent lysis of PRVinfected cells (49).Recently, several groups reported on the amino acid sequence motifs involved in the internalization of different alphaherpesvirus envelope glycoproteins. Two types...

Since the discovery of amphotericin B in 1955 the armamentarium of antimycotic drugs now embraces many new chemical classes: azoles, allylamines and candins. However, despite the wide variety in chemical structure, there is a lack of diversity in terms of mechanism of action. The mechanism of action of the main classes of antimycotics as well as the therapeutic value of some representatives is discussed. Some challenges to innovation will be highlighted that when overcome will herald more effective therapeutic interventions. Finally, we will list antimycotics that are at a late stage of development.

Viral glycoproteins gB and gD of the swine alphaherpesvirus pseudorabies virus (PRV), which is closely related to human herpes simplex virus and varicella-zoster virus, are able to drive internalization of antibodyantigen complexes that may form at the cell surface of infected monocytes, thereby protecting these cells from efficient antibody-mediated lysis. We found earlier that gB relies on an endocytosis motif in its cytoplasmic domain for its function during this internalization process. Here, we report that the PRV gD protein also contains a functional endocytosis motif (YRLL) in its cytoplasmic domain that drives spontaneous endocytosis of gD from the cell surface early in infection and that acts in concert with the endocytosis motif in gB to contribute to efficient internalization of antibody-antigen complexes in PRV-infected monocytes.Alphaherpesviruses have developed numerous strategies to delay or avoid recognition and elimination by different components of the immune system (11,21,45). The swine alphaherpesvirus pseudorabies virus (PRV) especially excels at circumventing antibody-dependent immunity, which allows it to replicate and sometimes spread in pigs that have been vaccinated with an inactivated vaccine (26, 48). PRV-infected blood monocytes play a pivotal role in spread of PRV in the presence of virus-neutralizing antibodies and carry the virus via the blood throughout the body (26).In PRV-infected blood monocytes, like in other PRV-infected cells, newly produced viral envelope proteins are incorporated in the plasma membrane (12,24), thereby rendering the cell recognizable for antibody-dependent immunity (13).However, we found earlier that binding of virus-specific antibodies to viral cell surface proteins in PRV-infected blood monocytes leads to rapid internalization of the antibody-antigen complexes (12), thereby lowering the susceptibility of the infected cell towards antibody-mediated cell lysis (41). This internalization process was found to be clathrin mediated and to depend on two of the PRV proteins at the cell surface, gB and gD (42).Clathrin-mediated endocytosis of cellular transmembrane proteins typically depends on so-called endocytosis motifs in their cytoplasmic domain, most notably YXXL and LL motifs (Y standing for tyrosine, L for leucine, and X for any amino acid). These motifs initiate endocytosis by establishing an interaction with the clathrin-associated AP-2 adaptor complex as a first step in the formation of clathrin-coated vesicles (3,4,20,36).We found that the function of gB in internalization of antibody-antigen complexes from the surface of PRV-infected monocytes depends on a functional tyrosine-based endocytosis motif (YQRL) in its cytoplasmic domain (10), and this motif was found to allow an interaction between gB and the AP-2 complex (43).How PRV gD is involved in internalization of antibodyantigen complexes, on the other hand, is unknown. PRV gD is a type I membrane glycoprotein of 402 amino acids, consisting of an extracellular domain, transmembrane region, and a ...

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