Bovine herpesvirus 1 (BHV-1) is a member of the Alphaherpesvirinae subfamily and is the prototype ruminant herpesvirus. BHV-1 causes a number of complications in cattle including upper respiratory tract disorders, conjunctivitis, genital disorders, abortions, and immune suppression. Like all herpesviruses, reactivation from latency can occur throughout the animal’s life. Of particular economic importance is the bovine respiratory disease complex (BRDC) or ‘shipping fever’, in which BHV-1 plays a major role. BRDC is an enormous economic concern as it costs the US cattle industry approximately one billion dollars annually. In order to generate improved gene-deleted vaccines against BHV-1, there is a need to understand the contributions of viral gene products during infection. US3 is a serine/threonine kinase present in BHV-1 and is thought to play major roles during viral infection. As in other herpesviruses, US3 in BHV-1 is expected to phosphorylate several cellular and/or viral proteins. We recently presented evidence that BHV-1 US3 phosphorylates both VP8 and VP22; however, further functional characteristics of BHV-1 US3 during viral infection have not been elucidated. The hypothesis of this project is that the deletion of the US3 gene leads to reduced BHV-1 fitness. To explore this hypothesis, we generated a US3-deleted (ΔUS3) and subsequent US3-rescued (US3R) BHV-1 virus. Using these viral mutants, we characterized the growth properties of the viruses, evaluated the effect of the US3 deletion on major structural BHV-1 proteins, characterized the protein composition of the mature virions, and, identified viral processes that were impaired in the deletion mutant. Initially, the ∆US3 virus was generated through a 3-step PCR strategy which replaced the gene of interest with an antibiotic resistance cassette. Following this, the US3 gene was rescued via a two-step en passant mutagenesis strategy which has been previously used to generate insertions, deletions, and substitutions in herpesvirus-containing bacterial artificial chromosome (BAC) DNA. In vitro characterization of ∆US3 BHV-1 has demonstrated that US3 deletion affects BHV-1 growth characteristics, expression kinetics of major structural proteins, mature virion composition, cell to cell spread, and the subcellular localization of key viral proteins during infection. Growth kinetics of ∆US3 BHV-1 were impaired compared to wild-type (WT) BHV-1, especially at late times post-infection. Plaque sizes formed by ∆US3 BHV-1 were significantly smaller than those formed by either WT or US3R BHV-1, demonstrating that US3 is important for cell to cell spread. The expression kinetics of major structural and regulatory BHV-1 proteins were different between cells infected with ∆US3 or WT BHV-1, and incorporation of these proteins into the mature viruses differed, demonstrating that US3 is instrumental in ensuring proper protein expression and mature virus composition in vitro. Of particular importance, glycoprotein B (gB), was shown to be expressed in higher quantities earlier during infection in the absence of US3, and that this protein was incorporated in significantly higher amounts in mature virions which lacked US3. Qualitative analysis of ∆US3 BHV-1 infected monolayers suggested the abolishment of cell to cell projections characteristic of WT BHV-1 infection. Finally, the disruption of gB in ∆US3 BHV-1 infected cells was confirmed by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Through confocal microscopy, evidence was provided that infection with ∆US3 BHV-1 possibly results in earlier expression of gB on the surface of cells and less intracellular accumulation of this protein during late stages of infection. The observed effect on the localization of intracellular gB in ∆US3 BHV-1 infected cells was quantified by flow cytometry. ∆US3 BHV-1 infected cells had approximately 25% higher gB expression on the surface of cells and a corresponding 25% decrease in intracellular gB. Although these differences have not yet been demonstrated to be statistically significant and not confirmed through infection with US3R BHV-1, this suggests that US3 may influence the synthesis and cellular trafficking of gB in vitro.

Herpes B virus, otherwise known as Macacine herpesvirus 1, is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplex, and is closely related to human herpes simplex viruses 1 and 2 (HSV1 and HSV2). B virus is endemic in macaque monkeys, but is capable of zoonotic transmission to humans resulting in fatality in greater than 80% of untreated cases. The goal of our lab is to understand the disparity in the outcome of infection between the natural host- macaques and the foreign host- humans. An important barrier to progress is the lack of understanding of host cell: B virus interactions in response to infection. An important pathway activated by stress, known as the mitogen activated protein kinase (MAPK) p38 pathway, is activated by B virus infection. Of particular interest is its role in regulating cellular translation via stimulation of activation of the eukaryotic initiation factor 4E (eIF4E). The activation of eIF4E is a vital rate-limiting step in translation, which can be manipulated by a variety of viruses. For example HSV1 can activate eIF4E through the p38 pathway but in the absence of this pathway eIF4E activity and viral titers are decreased. Because of the effect HSV1 has on the p38 pathway, and because B virus is a close relative of HSV1, we hypothesized that B virus also utilizes the p38 pathway to activate eIF4E in a host-dependent manner. In this dissertation, we show that the role of MAPK p38 with regard to translation is crucial to cellular processes that reduce virus replication in natural host cells, but within human cells this stress pathway appears not to play a role in reducing B virus replication. Data generated for this dissertation suggest that the p38 pathway is responsible in part for controlling the virus infection and spread within the natural host, but does not dampen virus replication in human host cells encountering the virus. Taken together, our results suggest that this pathway has at least one host-specific defense to combat B virus infection and that both cellular and viral proteins require the presence or absence of this pathway to function.

No description available.

In response to viral infection, host cells activate various antiviral defence mechanisms to inhibit virus replication. Therefore in order for a virus to replicate efficiently, it must counteract cellular antiviral defence. Promyelocytic leukemia protein (PML) is a cellular protein involved in intrinsic immunity. It inherently forms nuclear bodies (PML-NBs) that assemble at the site of viral genomes. Proteins related to epigenetic regulation are recruited to PML-NBs, and silence viral gene transcription. This study focuses on the role of two herpes simplex virus type 2 (HSV-2) proteins, ICP0 and Us3, in disrupting PML-NBs and counteracting cellular antiviral defence. En passant mutagenesis was used to create recombinant HSV-2 viruses lacking ICP0, Us3, or both ICP0 and Us3. Growth analysis of these recombinants indicates no growth defects for the ICP0Δ virus, while the Us3Δ virus grows to one log lower titres than wild type virus (WT). By contrast, the ICP0Δ virus displays a delay in PML-NB disruption, but the Us3Δ virus is as efficient as WT. However, Us3 is still important for PML-NB disruption, since the ICP0Δ/Us3Δ double mutant exhibits a greater delay than the ICP0Δ single mutant. Although PML is a mediator of the interferon (IFN) response and it was predicted that ICP0 and Us3 interfere with the IFN response through disruption of PML-NBs, my results show that only some HSV-2 Us3Δ clones are hypersensitive to the effects IFN, and others are resistant. Us3 affects more than one cellular pathway, and those cellular pathways are affected by more than one viral protein. I conclude that the activities of multiple viral proteins create a fine balance between activating cellular pathways to promote virus replication, and inhibiting cellular antiviral defence. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-01-23 10:55:16.715

No description available.

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection. Recently, work done in our laboratory determined that Us3 orthologues from herpes simplex type 2 (HSV-2) and pseudorabies virus (PRV) are capable of disrupting promyelocytic leukaemia (PML) protein nuclear bodies (-NBs). PML-NBs are discrete, dynamic nuclear bodies named for PML, their essential structural component and one that plays a key role in diverse cellular processes, including transcriptional regulation, apoptosis, and cellular antiviral defense. In infected cells, PML-NBs exert transcriptional silencing on the viral genome to prevent viral gene expression and virus replication. Based on this finding, my studies were aimed to understand the mechanism and physiological function of Us3-mediated PML-NB disruption. The degradation of one or more cellular proteins seems necessary for this Us3 activity, as the proteasome inhibitor, MG132, dramatically reduced Us3-mediated PML-NB disruption. The target of this proteasome activity is not likely PML protein, as Us3 expression did not lead to detectable PML protein degradation. Nonetheless, the involvement of proteasome activity suggests that Us3 may utilize the host ubiquitylation pathway to disrupt PML-NBs. Supporting this hypothesis, PRV and HSV-2 Us3 orthologues were shown to interact with KLHL21, a substrate adaptor protein for cullin-3 ubiquitin ligase. PRV and HSV-2 Us3 were re-localized to PML-NBs when co-expressed with KLHL21, and knock-down of KLHL21 prevented Us3-mediated PML-NB disruption. Taken together, these findings suggest that Us3-KLHL21 complex recruits the cullin-3 ubiquitin ligase to PML-NBs, where subsequent ubiquitylation of unknown target(s) leads to PML-NB disassembly. Since it is well established that PML is an important antiviral effector induced by interferon (IFN), Us3 may contribute to viral resistance to IFN by disrupting PML-NBs. Favoring this hypothesis, virus yield and viral gene transcription were dramatically reduced in IFN-exposed cells in the absence of Us3. These reductions were associated with an increased number of PML-NBs in the absence of Us3, and were partially recovered in cells knocked down for PML. Therefore, by disrupting PML-NBs, Us3 may alleviate IFN-induced, host-mediated transcriptional silencing of the viral genome, allowing efficient viral gene transcription and replication in cells exposed to IFN. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2014-04-28 16:36:54.079

Abstract Herpes simplex virus type 1 (HSV-1) is a common pathogen with an age-standardized seroprevalence of 52% in Finland. The most common manifestation of HSV-1 infection is labial herpes, but recently HSV-1 has emerged as the most common cause of primary genital herpes in Finnish women. HSV-1 can also lead to severe conditions such as encephalitis. After the primary lytic HSV-1 infection at the epithelia, the progeny viruses infect the innervating sensory neurons. The neuronal infection may lead to a quiescent infection form, called latency. Periodically, the virus may reactivate, which can lead to recurrent infection at the epithelia. During different phases of the viral life cycle the host cells try to restrict the infection. This study set out to investigate the roles of two HSV-1 proteins, γ134.5 and Us3 during different phases of the HSV-1 life cycle. The aim of the first study was to investigate how the deletion of Us3 affected host responses, especially Toll-like Receptor (TLR) signaling, in monocytic U937 cells. TLR3 expression was increased during Us3 deletion virus infections. This also led to increased activation of IRF-3 and increased expression of type I interferons (IFN) and an interferon stimulated protein. This study shows that TLR3 is involved in controlling the HSV-1 infection and that Us3 regulates IRF-3 activation. The second study focused on the role of the γ134.5 protein in HSV-1 latency. Embryonic mouse dorsal root ganglion (DRG) cultures were used as a cell culture model for HSV-1 latency and reactivation. In this model γ134.5 deletion viruses did not reactivate as efficiently as wild-type viruses, even though they replicated well and established latency in the neurons. Stress granules are part of the host response. In the third study, the roles of the innate immunity effectors HSV-1 Us3 and human Z-DNA binding protein 1 (ZBP1) in stress granule formation (SG) were studied. Wild-type HSV-1 efficiently prevented the formation of SGs. The overexpression of ZBP1 resulted in accumulation of smaller but more abundant SGs during oxidative stress. Overexpression of Us3 did not significantly affect the size or number of SGs, but during Us3 deletion virus infection, SG proteins localized to cis-Golgi. This work shows that HSV-1 uses Us3 to evade and modulate host responses and that the γ134.5 protein is required for reactivation in mouse DRG cultures. / Tiivistelmä Herpes simplex virus tyyppi 1 (HSV-1) on yleinen taudinaiheuttaja, jonka ikävakioitu seroprevalenssi Suomessa on 52 %. HSV-1 tunnetaan yleisimmin huuliherpeksen aiheuttajana, mutta myös kasvava osuus genitaaliherpeksistä on HSV-1:n aiheuttamia. HSV-1 voi johtaa myös vakaviin ilmentymiin, kuten aivotulehdukseen. Epiteelisolujen infektion tuottamia viruksia siirtyy aluetta hermottaviin tuntohermosoluihin, mikä voi johtaa piilevään infektiomuotoon eli latenssiin. Latentti virus voi ajoittain reaktivoitua, mistä voi seurata uusintainfektio. Isäntäsolu pyrkii rajoittamaan infektiota sen eri vaiheissa. Tämän tutkimuksen tarkoituksena oli selvittää kahden HSV-1:n virulenssiproteiinin, γ134.5:n ja Us3:n, merkitystä HSV-1:n elinkierrossa. Osatyössä I tutkittiin, miten Us3:n poisto vaikuttaa luontaisen immuniteetin vasteisiin, keskittyen etenkin Tollin kaltaisten reseptorien (TLR) signaalivälitykseen U937-monosyyttisoluissa. Us3-poistogeenisillä viruksilla suoritetuissa infektioissa TLR3:n ilmentyminen lisääntyi merkittävästi. Tämä johti myös lisääntyneeseen IRF-3-aktivaatioon sekä tyypin I interferonien ja interferonistimuloituvan proteiinin lisääntyneeseen ilmentymiseen. Tämä osoittaa, että TLR3 osallistuu HSV-1-viruksen tunnistukseen ja että Us3 säätelee IRF-3:n aktivaatiota. Osatyössä II keskityttiin γ134.5-proteiinin merkitykseen HSV-1:n latenssissa. Hiirialkioiden takajuuren hermoganglioita käytettiin soluviljelymallina HSV-1:n latenssin ja reaktivaation tutkimisessa. Tässä mallissa γ134.5-poistogeeniset virukset kasvoivat hyvin ja asettuivat latenteiksi, mutta eivät silti reaktivoituneet kuten luonnonkannan virukset. Stressijyväset ovat osa luontaista immuniteettia. Osatyössä III määritettiin HSV-1:n Us3-proteiinin ja ihmisen Z-DNA:han sitoutuvan proteiini 1:n (ZBP1) merkitystä stressijyvästen muodostumisessa. Luonnonkannan virus kykeni tehokkaasti estämään jyvästen muodostumisen. ZBP1:n yli-ilmentäminen oksidatiivisen stressin aikana johti suureen määrään pienikokoisia stressijyväsiä. Us3:n yli-ilmentäminen ei vaikuttanut stressijyväsiin, kun taas Us3-poistogeenisellä viruksella suoritetuissa infektioissa stressijyväsproteiinit paikantuivat Golgin laitteeseen. Tämä tutkimus osoittaa, että HSV-1 käyttää Us3-proteiinia luontaisten immuunivasteiden muunteluun ja että γ134.5-proteiini on välttämätön reaktivaatiossa hiiren hermoganglioissa.