RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1ß) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1ß, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1ß. The nsp1ß L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1ß-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-α/ß), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1ß, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1ß protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1ß binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1ß binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.
Assuntos
Evasão da Resposta Imune/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteínas não Estruturais Virais/imunologia , Replicação Viral/imunologia , Animais , Células HeLa , Humanos , SuínosRESUMO
UNLABELLED: Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection, leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport, the mechanisms and consequences of Nup98 proteolysis were examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2A(pro)). Incubation of bacterially expressed or in vitro-translated Nup98 with 2A(pro) results in proteolytic cleavage at multiple sites in vitro, indicating that 2A(pro) cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2A(pro) proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains the overall NPC structure and leaves other transport pathways unaffected. IMPORTANCE: Enteroviruses are dependent upon host nuclear RNA binding proteins for efficient replication. This study examines the mechanisms responsible for alterations in nuclear transport in enterovirus-infected cells that lead to the cytoplasmic accumulation of these proteins. The results demonstrate that the enterovirus 2A protease directly cleaves the nuclear pore complex (NPC) protein, Nup98, at amino acid positions G374 and G552 both in vitro and in infected cells. Cleavage at these positions results in the selective removal of the FG-containing N terminus of Nup98 from the NPC, while the C terminus remains associated. Nup88, a core component of the NPC that serves as a docking site for the C terminus of Nup98, remains associated with the NPC in infected cells. These findings help to explain the alterations in permeability and nuclear transport in enterovirus-infected cells and how NPCs remain functional for certain trafficking pathways despite significant alterations to their compositions.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Cisteína Endopeptidases/metabolismo , Enterovirus/enzimologia , Modelos Genéticos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Virais/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Mutagênese , Mutagênese Sítio-Dirigida , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-ß. In this study the impact of poliovirus infection on the type I interferon response has been examined. METHODS: The type I IFN response was assessed by measuring IFN-ß mRNA levels using qRT-PCR and normalizing to levels of ß-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR. RESULTS: The results show that poliovirus infection results in induction of very low levels of IFN-ß mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-ß mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed. CONCLUSIONS: Collectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , RNA Helicases DEAD-box/metabolismo , Interações Hospedeiro-Patógeno , Fator Regulador 3 de Interferon/antagonistas & inibidores , Interferon beta/antagonistas & inibidores , Poliovirus/imunologia , Poliovirus/fisiologia , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Immunoblotting , Helicase IFIH1 Induzida por Interferon , Interferon beta/biossíntese , Interferon beta/genética , Microscopia de Fluorescência , Proteólise , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A(pro)) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A(pro) is a major contributor to Nup62 processing. The ability of purified 2A(pro) to cleave bacterially expressed and purified Nup62 demonstrated that 2A(pro) directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A(pro) in vitro. This analysis revealed that 2A(pro) cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.
Assuntos
Núcleo Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poliomielite/enzimologia , Poliovirus/enzimologia , Rhinovirus/enzimologia , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/virologia , Cisteína Endopeptidases/genética , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Proteínas Virais/genéticaRESUMO
Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Poliovirus/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP27/fisiologia , Células HeLa , Proteínas de Choque Térmico , Resposta ao Choque Térmico , Humanos , Chaperonas Moleculares , Fosforilação , Proteínas de Ligação a Poli(A)/análise , Proteínas de Ligação a Poli(A)/fisiologia , RNA Mensageiro/biossíntese , Antígeno-1 Intracelular de Células T , Transcrição GênicaRESUMO
Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2A(pro) to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poliovirus/crescimento & desenvolvimento , Citoplasma/química , Citoplasma/metabolismo , Citoplasma/virologia , Células HeLa , Humanos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/análise , Poliovirus/metabolismo , RNA Mensageiro/metabolismoRESUMO
Laboratories working with closely related viruses need simple and cost-effective ways to rapidly validate viral stocks, detect contamination and measure the abundance of viral RNA species. Using RT-PCR and specific primers an approach for the specific detection of rhinovirus type 14 (RV14) or poliovirus type 1 (PV1) is presented. It is demonstrated that viral sequences can be amplified directly from viral stocks or from infected cells. In addition, the utility of this protocol for the detection of low levels of contaminating PV1 in RV14 stocks is shown. Further, using quantitative real-time PCR It is shown that this approach can be used for the quantitative analysis of viral RNA and replication kinetics in infected cells. This method should be useful for laboratories working with PV and RV14 and could be adapted easily for use by laboratories working with other rhinovirus and enterovirus serotypes.
Assuntos
Poliovirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rhinovirus/isolamento & purificação , Virologia/métodos , Sequência de Bases , Primers do DNA/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Poliovirus/genética , Rhinovirus/genéticaRESUMO
BACKGROUND: Solid nanostructures are versatile platforms for constructing hybrid drug delivery systems that have tremendous potential for improving disease prevention and treatment. The rationale and application of solid nanostructures in the context of drug delivery are explored in this article. OBJECTIVE: The purpose of this paper is to provide a concise review of the major attributes of solid nanostructures as they relate to drug delivery and to describe the outstanding issues that need to be addressed in order to develop these materials into clinically useful reagents. METHODS: The scope of this opinion has been restricted to solid nanostructures, where solid nanostructures are defined as those that are not biodegradable. The opinion has been further limited to the three primary types of nanostructures: nanoparticles, nanowires and nanotubes. RESULTS/CONCLUSION: There is a need for cross-disciplinary training and standardized protocols for developing and evaluating the efficacy of solid nanomaterials.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas , Animais , Carbono , Química Farmacêutica , Excipientes , Humanos , Nanofios , Tamanho da PartículaRESUMO
The non-toxic enzymic A subunit of Shiga toxin 1 (StxA1) reduces expression and replication of the bovine retroviruses, bovine leukemia virus and bovine immunodeficiency virus (BIV). Here, the impact of StxA1 on representative positive and negative stranded RNA viruses was compared. BIV and equine infectious anemia virus were sensitive to picomolar concentrations of StxA1 while poliovirus, rhinovirus, and vesicular stomatitis virus were only marginally sensitive to nanomolar concentrations of toxin. Thus, the length of the reproductive cycle and/or other factors, but not viral encapsulation may play a role in determining sensitivity to StxA1. The effects of StxA1 at concentrations from 0.01 to 10 microg/ml on the most sensitive virus (BIV-infected cultures of fetal bovine lung cells) were analyzed by electron microscopy 48 h post challenge. Cells treated with 0.1 microg StxA1/ml or higher toxin concentrations were similar in appearance and showed progressively fewer viral factories with increasing toxin concentration. However, cells treated with 0.01 microg/ml StxA1 had a radically different appearance, exhibiting smooth cell membranes and high vacuolization. These results showed that complex retroviruses were more sensitive to StxA1 than single-stranded RNA viruses and that StxA1 interfered with retroviral replication in a concentration-dependent manner.
Assuntos
Antivirais/farmacologia , Toxina Shiga I/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Imunodeficiência Bovina/efeitos dos fármacos , Vírus da Imunodeficiência Bovina/fisiologia , Testes de Sensibilidade Microbiana , Toxina Shiga I/química , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
Analysis of virus-host interactions has revealed a variety of ways in which viruses utilize and/or alter host functions in an effort to facilitate efficient replication. Recent work has suggested that certain RNA viruses that replicate in the cytoplasm disrupt the normal trafficking of cellular RNAs and proteins within the host cell. This review will examine the recent evidence showing that poliovirus and vesicular stomatitis virus (VSV) can inhibit nucleo-cytoplasmic transport within cells. Interestingly, the data indicate that inhibition by both viruses involves targeting components of the nuclear pore complex (NPC). Following this, several possible explanations for why viruses might disrupt nucleo-cytoplasmic transport are discussed. Finally, the possibility that disruption of nucleo-cytoplasmic trafficking may be a more common feature of RNA virus-host interactions than previously thought is examined.
Assuntos
Citoplasma/metabolismo , Poro Nuclear/metabolismo , Poliovirus/patogenicidade , Vírus da Estomatite Vesicular Indiana/patogenicidade , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Células HeLa , HumanosRESUMO
Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFN-competent mice.
Assuntos
Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/imunologia , Theilovirus/imunologia , Theilovirus/fisiologia , Proteínas Virais/imunologia , Proteínas Virais/fisiologia , Animais , Dimerização , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , RNA Mensageiro/metabolismoRESUMO
The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-kappaB, IRF-3 and ATF-2 which in turn activate transcription from the IFN-beta promoter. Here we have examined the type I interferon response in rhinovirus type 14-infected A549 cells, with particular emphasis on the status of the transcription factor IRF-3. Our results indicate that although rhinovirus type 14 (RV14) infection induces the activation of NF-kappaB and ATF-2, only very low levels of IFN-beta mRNA are detected. Analysis of ISG54 mRNA levels revealed very little induction of this IRF-3 responsive transcript and suggested that IRF-3 activation might be impaired. Examination of IRF-3 in RV14-infected cells demonstrated only low levels of phosphorylation, a lack of homodimer formation and an absence of nuclear accumulation indicating that this transcription factor is not activated. Inhibition of viral protein synthesis following infection resulted in an increase in IFN-beta mRNA levels indicating that viral gene products prevent induction of this pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.
Assuntos
Células Epiteliais/virologia , Fator Regulador 3 de Interferon/antagonistas & inibidores , Interferon Tipo I/antagonistas & inibidores , Alvéolos Pulmonares/citologia , Rhinovirus/patogenicidade , Linhagem Celular , Humanos , Fator Regulador 3 de Interferon/genética , Interferon Tipo I/genética , Interferon beta/antagonistas & inibidores , Interferon beta/genética , Interferon beta/metabolismo , Alvéolos Pulmonares/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rhinovirus/classificaçãoRESUMO
The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-kappaB, interferon regulatory factor 3 (IRF-3), and ATF-2, which in turn activate transcription from the IFN-beta promoter. Synthesis and subsequent secretion of IFN-beta activate the Jak/STAT signaling pathway, resulting in the transcriptional induction of the full spectrum of antiviral gene products. We utilized high-density microarrays to examine the transcriptional response to rhinovirus type 14 (RV14) infection in HeLa cells, with particular emphasis on the type I interferon response and production of IFN-beta. We found that, although RV14 infection results in altered levels of a wide variety of host mRNAs, induction of IFN-beta mRNA or activation of the Jak/STAT pathway is not seen. Prior work has shown, and our results have confirmed, that NF-kappaB and ATF-2 are activated following infection. Since many viruses are known to target IRF-3 to inhibit the induction of IFN-beta mRNA, we analyzed the status of IRF-3 in infected cells. IRF-3 was translocated to the nucleus and phosphorylated in RV14-infected cells. Despite this apparent activation, very little homodimerization of IRF-3 was evident following infection. Similar results in A549 lung alveolar epithelial cells demonstrated the biological relevance of these findings to RV14 pathogenesis. In addition, prior infection of cells with RV14 prevented the induction of IFN-beta mRNA following treatment with double-stranded RNA, indicating that RV14 encodes an activity that specifically inhibits this innate host defense pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.
Assuntos
Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Rhinovirus/fisiologia , Fator 2 Ativador da Transcrição/metabolismo , Linhagem Celular , Dimerização , Células HeLa , Humanos , Interferon Tipo I/antagonistas & inibidores , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/genética , Janus Quinase 1 , NF-kappa B/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro , Rhinovirus/patogenicidade , Fatores de Transcrição STAT/metabolismoRESUMO
The adenovirus IVa2 and L1 52/55-kDa proteins are involved in the assembly of new virus particles. Both proteins bind to the packaging sequence of the viral chromosome, and the lack of expression of either protein results in no virus progeny: the absence of the L1 52/55-kDa protein leads to formation of only empty capsids, and the absence of the IVa2 protein results in no capsid assembly. Furthermore, the IVa2 and L1 52/55-kDa proteins interact with each other during adenovirus infection. However, what is not yet clear is when and how this interaction occurs during the course of the viral infection. We defined the domains of the L1 52/55-kDa protein required for interaction with the IVa2 protein, DNA binding, and virus replication by constructing L1 52/55-kDa protein truncations. We found that the N-terminal 173 amino acids of the L1 52/55-kDa protein are essential for interaction with the IVa2 protein. However, for both DNA binding and complementation of the pm8001 mutant virus, which does not express the L1 52/55-kDa protein, the amino-terminal 331 amino acids of the L1 52/55-kDa protein are necessary. These results suggest that the production of infectious virus particles depends on the ability of the L1 52/55-kDa protein to bind to DNA.
Assuntos
Adenovírus Humanos/fisiologia , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Adenovírus Humanos/genética , Western Blotting , Fracionamento Celular , Linhagem Celular , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Humanos , Imunoprecipitação , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Deleção de Sequência , Proteínas Virais/genéticaRESUMO
Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/fisiologia , Rhinovirus/patogenicidade , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Autoantígenos/fisiologia , Citoplasma/fisiologia , Células HeLa , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Rhinovirus/fisiologia , Ribonucleoproteínas/fisiologia , Proteína Sequestossoma-1 , Antígeno SS-BRESUMO
DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.