RESUMEN
Pseudorabies virus (PRV), a porcine alphaherpesvirus, causes neurological disorders and reproductive failure in swine. It is capable of avoiding host antiviral responses, resulting in viral latency in infected animals. The mechanisms by which many PRV proteins help the virus to evade immune surveillance are poorly understood. In this study, we found that the PRV protein kinase, UL13, inhibits the IFN-ß signaling pathway by targeting interferon regulatory factor 3 (IRF3) for ubiquitination and degradation. PRV with mutant of UL13 is impaired in its ability to hinder IRF3 and interferon-ß (IFN-ß) activation, and has significantly less pathogenesis in mice that wild-type PRV. Our findings reveal an as yet undescribed mechanism utilized by PRV to evade host immune responses. PRV UL13 is a potential target for attenuated vaccines and antiviral drugs.
Asunto(s)
Herpesvirus Suido 1/genética , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Animales , Línea Celular , Herpesvirus Suido 1/enzimología , Evasión Inmune , Factor 3 Regulador del Interferón/genética , Interferón beta/inmunología , Masculino , Proteínas Quinasas/genética , Seudorrabia/virología , Porcinos , Testículo/citología , Ubiquitinación , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky's disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-ß production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-ß secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS-STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS-STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-ß gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS-STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-ß gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS-STING-mediated IFN-ß production by phosphorylating IRF3.
Asunto(s)
Herpesvirus Suido 1/fisiología , Factor 3 Regulador del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Células A549 , Animales , Perros , Células HEK293 , Herpesvirus Suido 1/enzimología , Humanos , Interferón beta/metabolismo , Células de Riñón Canino Madin Darby , FosforilaciónRESUMEN
Alphaherpesviral ribonucleotide reductase (RNR) is composed of large (pUL39, RR1) and small (pUL40, RR2) subunits. This enzyme can catalyze conversion of ribonucleotide to deoxynucleotide diphosphates that are further phosphorylated into deoxynucleotide triphosphate (dNTPs). The dNTPs are substrates for de novo viral DNA synthesis in infected host cells. The enzymatic activity of RNR depends on association between RR1 and RR2. However, the molecular basis underlying alphaherpesviral RNR complex formation is still largely unknown. In the current study, we investigated the pseudorabies virus (PRV) RNR interaction domains in pUL39 and pUL40. The interaction of pUL39 and pUL40 was identified by co-immunoprecipitation (co-IP) and colocalization analyses. Furthermore, the interaction amino acid (aa) domains in pUL39 and pUL40 were mapped using a series of truncated proteins. Consequently, the 90-210 aa in pUL39 was identified to be responsible for the interaction with pUL40. In turn, the 66-152, 218-258 and 280-303 aa in pUL40 could interact with pUL39, respectively. Deletion of 90-210 aa in pUL39 completely abrogated the interaction with pUL40. Deletion of 66-152, 218-258 and 280-303 aa in pUL40 remarkably weakened the interaction with pUL39, whereas a weak interaction could still be observed. Amino acid sequence alignments showed that the interaction domains identified in PRV pUL39/pUL40 were relatively non-conserved among the selected RNR subunits in alphaherpesviruses HSV1, HSV2, HHV3(VZV), BHV1, EHV1 and DEV. However, they were relatively conserved among PRV, HSV1 and HSV2. Collectively, our findings provided some molecular targets for inhibition of pUL39-pUL40 interaction to antagonize viral replication in PRV infected hosts.
Asunto(s)
Herpesvirus Suido 1/enzimología , Subunidades de Proteína/química , Ribonucleótido Reductasas/química , Línea Celular , Células HEK293 , Humanos , Nucleotidasas/metabolismo , Alineación de Secuencia , Replicación ViralRESUMEN
Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from betaherpesviruses and gammaherpesviruses. pUS3 homologs are multifunctional and are involved in many processes, including modification of the cytoskeleton, inhibition of apoptosis, and immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM), pointing to a modulatory function for pUS3 during deenvelopment. The HSV and pseudorabies virus (PrV) US3 genes are transcribed into two mRNAs encoding two pUS3 isoforms, which have different aminoterminal sequences and abundances. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform, or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture, while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested but disclosed a more severe defect in Madin-Darby bovine kidney cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV.IMPORTANCE A plethora of substrates and functions have been assigned to the alphaherpesviral pUS3 kinase, including a role in nuclear egress. In PrV, two different pUS3 isoforms are expressed, which differ in size, abundance, and intracellular localization. Their respective role in replication is unknown, however. Here, we show that efficient nuclear egress of PrV requires the smaller isoform and intact kinase activity, whereas absence of the larger isoform has no significant effect on viral replication. Thus, there is a clear distinction in function between the two US3 gene products of PrV.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Herpesvirus Suido 1/enzimología , Proteínas Serina-Treonina Quinasas/química , Proteínas Virales/química , Animales , Apoptosis , Bovinos , Chlorocebus aethiops , Citoesqueleto/metabolismo , Genoma Viral , Herpesvirus Suido 1/fisiología , Riñón/citología , Mutación , Membrana Nuclear/metabolismo , Fenotipo , Isoformas de Proteínas , Conejos , Células Vero , Ensamble de VirusRESUMEN
Alphaherpesviruses that establish persistent infections rely partly on their ability to evade host antiviral responses, notably the type I interferon (IFN) response. However, the mechanisms employed by alphaherpesviruses to avoid this response are not well understood. Pseudorabies virus (PRV) is an economically important pathogen and a useful model system for studying alphaherpesvirus biology. To identify PRV proteins that antagonize type I IFN signaling, we performed a screen by using an IFN-stimulated response element reporter in the swine cell line CRL. Unexpectedly, we identified the dUTPase UL50 as a strong inhibitor. We confirmed that UL50 has the ability to inhibit type I IFN signaling by performing ectopic expression of UL50 in cells and deletion of UL50 in PRV. Mechanistically, UL50 impeded type I IFN-induced STAT1 phosphorylation, likely by accelerating lysosomal degradation of IFN receptor 1 (IFNAR1). In addition, this UL50 activity was independent of its dUTPase activity and required amino acids 225 to 253 in the C-terminal region. The UL50 encoded by herpes simplex virus 1 (HSV-1) also possessed similar activity. Moreover, UL50-deleted PRV was more susceptible to IFN than UL50-proficient PRV. Our results suggest that in addition to its dUTPase activity, the UL50 protein of alphaherpesviruses possesses the ability to suppress type I IFN signaling by promoting lysosomal degradation of IFNAR1, thereby contributing to immune evasion. This finding reveals UL50 as a potential antiviral target.IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that this alphaherpesvirus could utilize its encoded dUTPase UL50 to induce IFNAR1 degradation and inhibit type I IFN signaling in an enzymatic activity-independent manner. Our finding reveals a mechanism employed by an alphaherpesvirus to evade the immune response and indicates that UL50 is an important viral protein in pathogenesis and is a potential target for antiviral drug development.
Asunto(s)
Herpesvirus Suido 1/enzimología , Interferón Tipo I/farmacología , Lisosomas/metabolismo , Seudorrabia/metabolismo , Pirofosfatasas/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Células HeLa , Herpesvirus Suido 1/genética , Humanos , Evasión Inmune , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/virología , Fosforilación , Proteolisis , Seudorrabia/tratamiento farmacológico , Seudorrabia/virología , Pirofosfatasas/genética , Receptor de Interferón alfa y beta/genética , Homología de Secuencia , Transducción de Señal , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The processivity factors (PFs) of herpesviruses confer processivity to the DNA polymerase. Understanding whether the herpesvirus PFs function as monomers or multimers is important for clarifying the mechanism by which they provide the DNA polymerase with processivity. Herpes simplex virus type 1 UL42 is a monomer, whereas human cytomegalovirus UL44, Epstein-Barr virus BMRF1, and Kaposi's sarcoma-associated herpesvirus PF-8 exist as dimers. However, the oligomeric status of the pseudorabies virus (PRV) DNA polymerase PF UL42 has not been determined. Using fluorescence confocal microscopy and chemical crosslinking, we confirmed that UL42 is a monomer when expressed in vitro. Crosslinking of nuclear extracts from PRV-infected or uninfected PK-15 cells verified that UL42 exists as a monomer in vivo. Our demonstration that UL42 exists as a monomer in vitro and in vivo contributes to the further investigation of the mechanism used by UL42 to achieve processivity.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Suido 1/enzimología , Seudorrabia/virología , Enfermedades de los Porcinos/virología , Proteínas Virales/metabolismo , Animales , ADN Polimerasa Dirigida por ADN/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/metabolismo , Porcinos , Proteínas Virales/genéticaRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Herpesvirus Suido 1/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Seudorrabia/enzimología , Enfermedades de los Porcinos/enzimología , Proteínas Virales/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Herpesvirus Suido 1/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Seudorrabia/metabolismo , Seudorrabia/virología , Porcinos , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/virología , Proteínas Virales/genética , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genéticaRESUMEN
A 948-bp sequence of the UL2 gene was amplified from the pseudorabies virus (PRV) Becker strain genome using polymerase chain reaction, and the gene identity was confirmed through further cloning and sequencing. Bioinformatic analysis indicated that the PRV UL2 gene encodes a putative polypeptide with 315-amino acid residues. Its encoding protein, designated UL2, has a conserved uracil-DNA glycosylase (UDG)_F1 domain, which is closely related to the herpesvirus UDG family and is highly conserved among its counterparts encoded by UDG genes. Multiple nucleic acid and amino acid sequence alignments suggested that the product of PRV UL2 has a relatively higher homology with UL2-like proteins of Alphaherpesvirinae than that of other subfamilies of Herpesviridae. In addition, phylogenetic analysis showed that PRV UL2 had a close evolutionary relationship with members of Alphaherpesvirinae, especially members of the genus Varicellovirus of bovine herpesvirus 1 and bovine herpesvirus 5. Antigen prediction indicated the presence of several potential B-cell epitopes in PRV UL2. In addition, secondary structure and 3-dimensional structure prediction revealed that PRV UL2 consisted predominantly of an α-helix. Taken together, these results provide molecular biological insight for the further study of the function and mechanism of UL2 during PRV infection.
Asunto(s)
Herpesvirus Suido 1/genética , Uracil-ADN Glicosidasa/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Genes Virales , Herpesvirus Suido 1/enzimología , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Uracil-ADN Glicosidasa/química , Proteínas Virales/químicaRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Herpesvirus Suido 1/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Internalización del Virus , Western Blotting , Forma de la Célula/fisiología , Inhibidores Enzimáticos/metabolismo , Microscopía Fluorescente , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Quinasas p21 Activadas/antagonistas & inhibidoresRESUMEN
The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.
Asunto(s)
Alphaherpesvirinae/enzimología , Regulación de la Expresión Génica , Proteínas Asociadas a Matriz Nuclear/metabolismo , Sistemas de Lectura Abierta/fisiología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Alphaherpesvirinae/clasificación , Alphaherpesvirinae/genética , Alphaherpesvirinae/metabolismo , Línea Celular , Fibroblastos/virología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/metabolismo , Herpesvirus Humano 3/enzimología , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/metabolismo , Humanos , Riñón/citología , Riñón/virología , Proteínas Asociadas a Matriz Nuclear/genética , Fosforilación , Proteínas Quinasas/genética , Proteínas de Unión al ARN/genéticaRESUMEN
A serine/threonine (S/T) kinase encoded by the US3 gene of herpes simplex virus type 1 (HSV-1) is conserved in varicella-zoster virus (VZV) and pseudorabies virus (PRV). Expression of US3 kinase in cells transformed with US3 expression plasmids or infected with each virus results in hyperphosphorylation of histone deacetylase 2 (HDAC2). Mapping studies revealed that each US3 kinase phosphorylates HDAC2 at the same unique conserved Ser residue in its C terminus. HDAC2 was also hyperphosphorylated in cells infected with PRV lacking US3 kinase, indicating that hyperphosphorylation of HDAC2 by PRV occurs in a US3-independent manner. Specific chemical inhibition of class I HDAC activity increases the plaquing efficiency of VZV and PRV lacking US3 or its enzymatic activity, whereas only minimal effects are observed with wild-type viruses, suggesting that VZV and PRV US3 kinase activities target HDACs to reduce viral genome silencing and allow efficient viral replication. However, no effect was observed for wild-type or US3 null HSV-1. Thus, we have demonstrated that while HDAC2 is a conserved target of alphaherpesvirus US3 kinases, the functional significance of these events is virus specific.
Asunto(s)
Alphaherpesvirinae/enzimología , Histona Desacetilasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Alphaherpesvirinae/genética , Alphaherpesvirinae/patogenicidad , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Cartilla de ADN/genética , Genes Virales , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/genética , Herpesvirus Humano 3/enzimología , Herpesvirus Humano 3/genética , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Homología de Secuencia de Aminoácido , Células Vero , Proteínas Virales/genéticaRESUMEN
The neuroinvasive property of several alpha-herpesviruses underlies an uncommon infectious process that includes the establishment of life-long latent infections in sensory neurons of the peripheral nervous system. Several herpesvirus proteins are required for replication and dissemination within the nervous system, indicating that exploiting the nervous system as a niche for productive infection requires a specialized set of functions encoded by the virus. Whether initial entry into the nervous system from peripheral tissues also requires specialized viral functions is not known. Here we show that a conserved deubiquitinase domain embedded within a pseudorabies virus structural protein, pUL36, is essential for initial neural invasion, but is subsequently dispensable for transmission within and between neurons of the mammalian nervous system. These findings indicate that the deubiquitinase contributes to neurovirulence by participating in a previously unrecognized initial step in neuroinvasion.
Asunto(s)
Endopeptidasas/fisiología , Herpesvirus Suido 1/enzimología , Seudorrabia/virología , Células Receptoras Sensoriales/virología , Ubiquitina/metabolismo , Proteínas Estructurales Virales/fisiología , Animales , Cámara Anterior/virología , Transporte Axonal/fisiología , Chlorocebus aethiops , Endopeptidasas/genética , Infecciones Virales del Ojo/virología , Herpesvirus Suido 1/genética , Masculino , Seudorrabia/fisiopatología , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Proteasas Ubiquitina-Específicas , Células Vero , Proteínas Estructurales Virales/genéticaRESUMEN
Different viruses exploit the host cytoskeleton to facilitate replication and spread. The conserved US3 protein of the alphaherpesvirus pseudorabies virus induces actin stress fiber disassembly and formation of actin-containing cell projections, which are associated with enhanced intercellular virus spread. Proteins of members of other virus families, notably vaccinia virus F11L protein and human immunodeficiency virus Nef protein, induce actin rearrangements that are very similar to those induced by US3. Interestingly, unlike F11L and Nef, the US3 protein displays serine/threonine kinase activity. Here, we report that the kinase activity of pseudorabies virus US3 is absolutely required for its actin modulating activity. These data show that different viruses have developed independent mechanisms to induce very similar actin rearrangements.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Seudorrabia/patología , Proteínas Virales/metabolismo , Animales , Línea Celular , Citoesqueleto/enzimología , RatonesRESUMEN
Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses and constitute a major site of virus latency and reactivation. Earlier we showed that porcine TG neurons are remarkably more resistant towards (apoptotic) cell death resulting from infection by the swine alphaherpesvirus pseudorabies virus (PRV) compared to a broad range of other primary porcine cell types and that this resistance does not depend on the strongly anti-apoptotic US3 viral protein kinase (Geenen, K., Favoreel, H.W., Nauwynck, H.J., 2005a. Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared with other porcine cell types in vitro. J. Gen. Virol. 86, 1251-1260). Although other viral anti-apoptotic proteins may be involved in survival of TG neurons during PRV infection, an additional factor may be that TG neurons possess a cell type-dependent capacity to withstand apoptosis compared to other cell types. To investigate this, we treated uninfected porcine TG cultures, swine kidney cells, and porcine superior cervical ganglion (SCG) neurons with several apoptosis-inducing reagents (staurosporine, camptothecin and genistein). None of these reagents were able to trigger substantial apoptotic cell death in TG neurons, whereas non-neuronal TG cells, swine kidney cells, and SCG neurons showed a clear dose-dependent increase in apoptosis using either of these reagents. In conclusion, sensory TG neurons may contain a cell type-specific capacity to withstand different apoptotic assaults, including infection with an alphaherpesvirus.
Asunto(s)
Apoptosis , Inhibidores Enzimáticos/farmacología , Herpesvirus Suido 1/patogenicidad , Neuronas/virología , Seudorrabia/virología , Ganglio del Trigémino/virología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Camptotecina/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Genisteína/farmacología , Herpesvirus Suido 1/enzimología , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Estaurosporina/farmacología , Porcinos , Ganglio del Trigémino/citología , Ganglio del Trigémino/efectos de los fármacos , Latencia del VirusRESUMEN
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.
Asunto(s)
Citoesqueleto/ultraestructura , Citoesqueleto/virología , Herpesvirus Suido 1/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Testículo/ultraestructura , Amidas/farmacología , Animales , Cápside/efectos de los fármacos , Cápside/fisiología , Depsipéptidos/farmacología , Herpesvirus Suido 1/efectos de los fármacos , Herpesvirus Suido 1/ultraestructura , Masculino , Microscopía Confocal , Microtúbulos/ultraestructura , Microtúbulos/virología , Piridinas/farmacología , Porcinos , Testículo/virología , Ensayo de Placa ViralRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Herpesvirus Suido 1/fisiología , Proteínas Quinasas/fisiología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Expresión Génica , Herpesvirus Suido 1/enzimología , Isoenzimas/biosíntesis , Isoenzimas/fisiología , Masculino , Proteínas Quinasas/biosíntesis , Sorbitol/farmacología , Estaurosporina/farmacología , PorcinosRESUMEN
During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient transfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo.
Asunto(s)
Herpesvirus Suido 1/fisiología , Ribonucleasas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Herpesvirus Suido 1/enzimología , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Proteínas Recombinantes/metabolismo , Ribonucleasas/biosíntesis , Homología de Secuencia de Aminoácido , Transfección , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Replicación ViralRESUMEN
The Us3 protein kinase encoded by herpes simplex virus type-1 (HSV-1) suppresses apoptosis in infected cells and is sufficient to block apoptosis induced by overexpression of Bad [Proc. Natl. Acad. Sci. 98 (2001) 10410]. While Us3 can induce phosphorylation of Bad, phosphorylation of Bad is dispensable for Us3 anti-apoptotic function [J. Virol. 77 (2003) 6567]. We extend the findings with Bad to demonstrate that Us3 blocks apoptosis induced by overexpression of Bid, a factor parallel to Bad in the apoptotic pathway, and Bax, a factor downstream of Bad in the apoptotic pathway. A previous report suggested that Us3 exerts its effects at a premitochondrial stage [J. Virol. 75 (2001) 5491], but our results suggest that Us3 exerts anti-apoptotic effects downstream of the mitochondria. We show that the kinase activity of Us3 is necessary for Us3 anti-apoptotic effects, because a catalytically inactive form of Us3 was unable to block apoptosis. A second function of Us3, primary envelopment during viral egress, is conserved in the Us3 homologue of Pseudorabies virus (PRV) [J. Gen. Virol. 82 (2001) 2363]. Experiments published here demonstrate that PRV Us3 can also block apoptosis induced by Bax, suggesting that the anti-apoptotic activity of Us3 is conserved across alpha-herpesviruses.
Asunto(s)
Apoptosis , Herpesvirus Humano 1/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Línea Celular , Línea Celular Tumoral , Herpesvirus Humano 1/enzimología , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/fisiología , Humanos , Mitocondrias/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transfección , Proteínas Virales , Proteína X Asociada a bcl-2RESUMEN
Free-ranging feral swine (Sus scrofa) are known to be present in at least 32 states of the USA and are continuously expanding their range. Infection with pseudorabies virus (PRV) occurs in feral swine and the primary route of transmission in free-living conditions seems to be venereal. Between 1995 and 1999, naturally infected feral swine and experimentally infected hybrid progeny of feral and domestic swine, were kept in isolation and evaluated for occurrence of latent PRV indigenous to feral swine in sacral and trigeminal ganglia and tonsil. Sacral ganglia were shown, by polymerase chain reaction (PCR) amplification of the thymidine kinase (TK) gene of PRV, to be the most frequent sites of latency of PRV. Nine (56%) of 16 sacral ganglia, seven (44%) of 16 trigeminal ganglia, and five (39%) of 13 tonsils from naturally infected feral swine were positive for PCR amplification of TK sequences of PRV. These tissues were negative for PRV when viral isolation was attempted in Vero cells. DNA sequencing of cloned TK fragments from the sacral ganglia of two feral swine, showed only one nucleotide difference between the two fragments and extensive sequence homology to fragment sequences from various domestic swine PRV strains from China, Northern Ireland, and the USA. The hybrid feral domestic swine, experimentally inoculated with an indigenous feral swine PRV isolate by either the genital or respiratory route, acquired the infection but showed no clinical signs of pseudorabies. Virus inoculated into either the genital or respiratory tract could, at times, be isolated from both these sites. The most common latency sites were the sacral ganglia, regardless of the route and dose of infection in these experimentally infected hybrids. Nine of 10 sacral ganglia, six of 10 trigeminal ganglia, and three of 10 tonsils were positive for PCR amplification of TK sequences. No virus was isolated from these tissues in Vero cells. The demonstration of the sacral ganglia as the most common sites of latency of pseudorabies viruses indigenous to feral swine, supports the hypothesis that these viruses are primarily transmitted venereally, and not by the respiratory route as is common in domestic swine, in which the trigeminal ganglia are the predominant sites of virus latency.
Asunto(s)
Ganglios Simpáticos/virología , Herpesvirus Suido 1/fisiología , Seudorrabia/transmisión , Enfermedades de los Porcinos/transmisión , Latencia del Virus , Animales , Animales Domésticos , Animales Salvajes , Secuencia de Bases , ADN Viral/química , Femenino , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/genética , Masculino , Datos de Secuencia Molecular , Tonsila Palatina/virología , Reacción en Cadena de la Polimerasa/veterinaria , Seudorrabia/epidemiología , Seudorrabia/virología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/transmisión , Infecciones del Sistema Respiratorio/veterinaria , Infecciones del Sistema Respiratorio/virología , Homología de Secuencia de Ácido Nucleico , Enfermedades Virales de Transmisión Sexual/epidemiología , Enfermedades Virales de Transmisión Sexual/transmisión , Enfermedades Virales de Transmisión Sexual/veterinaria , Sus scrofa , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/virología , Timidina Quinasa/genética , Ganglio del Trigémino/virologíaRESUMEN
12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.