Most large DNA viruses, like herpesviruses, encode anti-apoptotic proteins to interfere with the apoptotic cellular response to infection. Previous studies have shown that the US3 protein kinase of herpes simplex virus, in contrast to US3 of bovine herpes virus 1, is very potent in protecting cells from apoptosis induced by the virus itself or by a broad range of exogenous apoptotic stimuli. Here, we demonstrate that US3 of the swine alphaherpesvirus pseudorabies virus (PRV) suppresses PRV-induced apoptosis in swine-testicle (ST) cells at late stages in infection, and that it protects ST cells from apoptosis induced by either sorbitol or staurosporine. Interestingly, PRV US3 encodes a short and a long isoform, the latter of which contains a functional mitochondrial localization sequence. Transient transfections showed that the PRV US3 long isoform is more efficient in protecting ST cells from PRV- or staurosporine-induced apoptosis, suggesting a potential advantage for the mitochondrial localization of PRV US3 in implementing its anti-apoptotic function.
The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/ threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.The alphaherpesviruses, such as the human pathogens herpes simplex virus types 1 and 2 (HSV-1; HSV-2), varicellazoster virus, and the agricultural-animal pathogens, such as pseudorabies virus (PRV), bovine herpesvirus type 1, and Marek's disease virus, are pantropic but are capable of invading the peripheral nervous systems (PNS) and central nervous systems (CNS) of susceptible animals (18,34,39). The infection of polarized cells, such as epithelial cells and neurons, plays a central role in the alphaherpesvirus life cycle. For example, primary infection usually occurs in mucosal epithelial cells, but after replication and release of virions at the basolateral surface, infection readily spreads to underlying nerve terminals of neurons of the PNS. The encapsidated viral genome then spreads directionally inside axons via microtubules to the cell bodies of sensory and autonomic ganglia. After entering the nucleus, the viral DNA is transcribed, and the neuron may experience a productive or latent infection. Most infections of natural hosts result in latent infections. After reactivation from a latent infection in PNS neurons, viral genomes move into axons and reverse their original direction by spreading back to the periphery, where upon release, progeny virions can infect epithelial cells and spread to other hosts. In rare cases after infection of the natural host, primary infection results in systemic spread of infection and occasional invasion of the CNS. When nonnatural hosts are infected, the outcome is reversed: latent infections and survival are rare, while rapid death and invasion of the CNS are common (3,5,25,35).It is well established for PRV that infection of nonnatural hosts, such as dogs, cattle, and rodents, by virulent strains causes a peripheral neuropathy characterized by violent pruritus (the mad itch) and rapid demise of the animal (51). Certain live, attenuated vaccine strains (notably the well-studied Bartha strain of PRV) have markedly re...
The herpesvirus family is defined by a common infection pattern and viral particle architecture. The virion is composed of three distinct structural entities. The exposed outer surface of the virion consists of a lipid bilayer derived from cellular membranes and contains a dense collection of viral transmembrane proteins. This surrounds an icosahedral capsid that carries the viral genome. Between the envelope and capsid lies a layer of virally encoded proteins collectively know as the tegument. The trafficking of the assembling particle through the cell during its acquisition of virion components defines the egress pathway. DNA filled capsids are assembled in the nucleus then bud into the inner nuclear envelope. During this primary envelopment step, virions acquire a layer of primary tegument and form a 'primary enveloped' intermediate in the perinuclear space. Via fusion with the outer nuclear envelope this particle de-envelops and is released into the cytoplasm. The acquisition of cytoplasmic tegument proteins occurs through a web of protein-protein interactions. Finally, the virion undergoes secondary envelopment upon budding into a trans-golgi like compartment containing viral membrane glycoproteins. Fusion of this compartment with the plasma membrane releases infectious particles into the extracellular environment.Alpha-herpesviruses, such as pseudorabies virus, encode a unique protein important in the primary envelopment steps of egress. This serine/threonine kinase, Us3, is non-essential in tissue culture but has a cell type dependent replication defect as well as an extracellular particle 'release' defect. Interestingly, Us3 is acquired in the nucleus and is one of the only tegument components found in both the primary and secondary tegument [1]. Us3 also orchestrates the localization of several viral proteins to the inner nuclear envelope and in PRV infected explants of nasal epithelia, Us3 mutants are impaired in the de-envelopment of primary enveloped particles in the perinuclear space [2,3]. In this study we examined infected cells at late times during infection to identify the possible roles of Us3 in virion assembly and egress. PK15 cells were infected for 16hours then scraped, pelleted, and processed for conventional transmission electron microscopy.Our results indicate that the peri-nuclear, de-envelopment delay previously seen in primary explant tissue also occurs in the porcine kidney tissue culture (PK15) cell line. Providing an easier culture system in which to investigate this phenotype. In addition, the secondary envelopment process in Us3null infections also appears compromised. In contrast to wildtype infection in which tegumented cytoplasmic particles readily line cytoplasmic vesicles and undergo secondary envelopment, Us3null particles appear disordered within the cytoplasm. These results indicate Us3 may not only facilitate de-envelopment in the primary stage of egress but also play a role in cytoplasmic tegument acquisition or targeting necessary for efficient secondary envelopment Re...
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