RESUMO
Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.
RESUMO
Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.
Assuntos
Cardiomiopatia Dilatada , Infecções por Coxsackievirus , Enterovirus Humano B , Humanos , Antígenos Virais , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/virologia , Infecções por Coxsackievirus/complicações , Enterovirus Humano B/metabolismo , Genômica , Miócitos Cardíacos/virologia , Peptídeo Hidrolases , Infecção Persistente , RNA Viral/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
BACKGROUND: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. METHODS: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. RESULTS: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. CONCLUSIONS: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.
Assuntos
Regiões 5' não Traduzidas/genética , Cardiomiopatia Dilatada/virologia , Cisteína Endopeptidases/genética , Enterovirus Humano B/isolamento & purificação , Miócitos Cardíacos/virologia , RNA Viral/genética , Proteínas Virais/genética , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/patologia , Células Cultivadas , Cisteína Endopeptidases/biossíntese , Efeito Citopatogênico Viral , DNA Complementar/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologia , Infecções por Enterovirus/complicações , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Miocardite/complicações , Miocardite/virologia , Deleção de Sequência , Transfecção , Proteínas Virais/biossíntese , Latência Viral , Replicação ViralRESUMO
Our understanding of gene expression has come far since the 'one-gene one-polypeptide' hypothesis proposed by Beadle and Tatum. In this review, we address the gradual recognition that a growing number of polycistronic genes, originally discovered in viruses, are being identified within the mammalian genome, and that these may provide new insights into disease mechanisms and treatment. We carried out a systematic literature review identifying 13 mammalian genes for which there is evidence for polycistronic expression via translation through an internal ribosome entry site (IRES). Although the canonical mechanism of translation initiation has been studied extensively, here we highlight a process of noncanonical translation, IRES-mediated translation, that is a growing source for understanding complex inheritance, the elucidation of disease mechanisms, and the discovery of novel therapeutic targets. Identification of additional polycistronic genes may provide new insights into disease therapy and allow for new discoveries of both translational and disease mechanisms.
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Doenças Genéticas Inatas/genética , Sítios Internos de Entrada Ribossomal/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Doenças Genéticas Inatas/terapia , Humanos , Mamíferos , Terapia de Alvo Molecular , RNA Mensageiro/biossínteseRESUMO
Due to the limiting coding capacity for members of the Picornaviridae family of positive-strand RNA viruses, their successful replication cycles require complex interactions with host cell functions. These interactions span from the down-modulation of many aspects of cellular metabolism to the hijacking of specific host functions used during viral translation, RNA replication, and other steps of infection by picornaviruses, such as human rhinovirus, coxsackievirus, poliovirus, foot-and-mouth disease virus, enterovirus D-68, and a wide range of other human and nonhuman viruses. Although picornaviruses replicate exclusively in the cytoplasm of infected cells, they have extensive interactions with host cell nuclei and the proteins and RNAs that normally reside in this compartment of the cell. This review will highlight some of the more recent studies that have revealed how picornavirus infections impact the RNA metabolism of the host cell posttranscriptionally and how they usurp and modify host RNA binding proteins as well as microRNAs to potentiate viral replication.
Assuntos
Interações Hospedeiro-Patógeno , Infecções por Picornaviridae/virologia , Picornaviridae/patogenicidade , RNA/metabolismo , Replicação Viral , Animais , Humanos , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/metabolismo , RNA/genéticaRESUMO
Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.
Assuntos
Núcleo Celular/fisiologia , Citoplasma/virologia , Interações Hospedeiro-Patógeno , Rhinovirus/fisiologia , Transporte Ativo do Núcleo Celular , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/metabolismo , Transporte Proteico , Proteólise , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5'-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5' stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5' deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5' deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7- and 21-nucleotide deletions. Since 5' stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CDpro, to deleted positive-strand RNAs corresponding to the 5' end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5'-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5' end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5'-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.
Assuntos
Enterovirus Humano B/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Deleção de Sequência , Replicação Viral , Animais , Células Cultivadas , Análise Mutacional de DNA , Enterovirus Humano B/genética , Humanos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/virologia , Ligação ProteicaRESUMO
Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a 'non-cleavable' bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free 'click' reaction, leading to a VPg-triazole-RNA construct that was 'non-cleavable' by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.
Assuntos
Peptídeos/química , Picornaviridae/genética , Biossíntese de Proteínas , RNA Viral/biossíntese , RNA Viral/química , Replicação Viral , Química Click , Enterovirus/genética , Genoma Viral , Células HeLa , Humanos , Picornaviridae/fisiologia , RNA/química , Proteínas Virais/químicaRESUMO
UNLABELLED: Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE: Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition.
Assuntos
Poliomielite/metabolismo , Poliovirus/genética , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Proteases Virais 3C , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Poliomielite/genética , Poliomielite/virologia , Poliovirus/enzimologia , Poliovirus/fisiologia , Processamento de Proteína Pós-Traducional , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein-RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5'-tyrosyl-DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein-RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host-pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.
Assuntos
Proteínas Nucleares/metabolismo , Picornaviridae/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Imunofluorescência , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Microscopia Confocal , Proteínas Nucleares/genética , Diester Fosfórico Hidrolases , Picornaviridae/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genéticaRESUMO
During infection by picornaviruses, the cellular environment is modified to favour virus replication. This includes the modification of specific host proteins, including the recently discovered viral proteinase cleavage of mRNA decay factor AU-rich binding factor 1 (AUF1). This cellular RNA-binding protein was shown previously to act as a restriction factor during poliovirus, rhinovirus and coxsackievirus infection. During infection by these viruses, AUF1 relocalizes to the cytoplasm and is cleaved by the viral 3C/3CD proteinase. In this study, we demonstrated that replication of encephalomyocarditis virus (EMCV), a picornavirus belonging to the genus Cardiovirus, is AUF1 independent. During EMCV infection, AUF1 relocalized to the cytoplasm; however, unlike what is seen during enterovirus infections, AUF1 was not cleaved to detectable levels, even at late times after infection. This suggests that AUF1 does not act broadly as an inhibitor of picornavirus infections but may instead act as a selective restriction factor targeting members of the genus Enterovirus.
Assuntos
Vírus da Encefalomiocardite/imunologia , Vírus da Encefalomiocardite/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Estabilidade de RNA , Replicação Viral , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , HumanosRESUMO
To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3' noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5' NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5' NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses.
Assuntos
Infecções por Coxsackievirus/virologia , Enterovirus/patogenicidade , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , Infecções por Picornaviridae/virologia , Estabilidade de RNA , Rhinovirus/patogenicidade , Proteases Virais 3C , Animais , Células Cultivadas , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/metabolismo , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Citoplasma/virologia , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/virologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Imunofluorescência , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Camundongos , Camundongos Knockout , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/metabolismo , Poliovirus/genética , Biossíntese de Proteínas , Isoformas de Proteínas , RNA não Traduzido/genética , RNA Viral/genética , Coelhos , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Infection of mammalian cells by picornaviruses results in the nucleocytoplasmic redistribution of certain host cell proteins. These viruses interfere with import-export pathways, allowing for the cytoplasmic accumulation of nuclear proteins that are then available to function in viral processes. We recently described the cytoplasmic relocalization of cellular splicing factor SRp20 during poliovirus infection. SRp20 is an important internal ribosome entry site (IRES) trans-acting factor (ITAF) for poliovirus IRES-mediated translation; however, it is not known whether other picornaviruses utilize SRp20 as an ITAF and direct its cytoplasmic relocalization. Also, the mechanism by which poliovirus directs the accumulation of SRp20 in the cytoplasm of the infected cell is currently unknown. Work described in this report demonstrated that infection by another picornavirus (coxsackievirus B3) causes SRp20 to relocalize from the nucleus to the cytoplasm of HeLa cells, similar to poliovirus infection; however, SRp20 is relocalized to a somewhat lesser extent in the cytoplasm of HeLa cells during infection by yet another picornavirus (human rhinovirus 16). We show that expression of poliovirus 2A proteinase is sufficient to cause the nucleocytoplasmic redistribution of SRp20. Following expression of poliovirus 2A proteinase in HeLa cells, we detect cleavage of specific nuclear pore proteins known to be cleaved during poliovirus infection. We also find that expression of human rhinovirus 16 2A proteinase alone can cause efficient cytoplasmic relocalization of SRp20, despite the lower levels of SRp20 relocalization observed during rhinovirus infection compared to poliovirus. Taken together, these results further define the mechanism of SRp20 cellular redistribution during picornavirus infections, and they provide additional insight into some of the differences observed between human rhinovirus and other enterovirus infections.
Assuntos
Cisteína Endopeptidases/metabolismo , Infecções por Picornaviridae/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Infecções por Coxsackievirus/metabolismo , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Citoplasma/metabolismo , Enterovirus Humano B/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Poliovirus/metabolismo , Rhinovirus/metabolismo , Fatores de Processamento de Serina-Arginina , Transativadores/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a â¼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions.
Assuntos
Núcleo Celular/metabolismo , Modelos Biológicos , Poliomielite/metabolismo , Poliovirus/metabolismo , Biossíntese de Proteínas , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular/genética , Motivos de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/virologia , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/virologia , Células HeLa , Humanos , Mutação , Poliomielite/genética , Poliovirus/genética , Ligação Proteica , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-ArgininaRESUMO
SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.
Assuntos
Regulação Viral da Expressão Gênica/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas não Estruturais Virais/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Moldes Genéticos , Proteínas não Estruturais Virais/genéticaRESUMO
Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.
Assuntos
Grânulos de Estresse , Viroses , Humanos , DNA Helicases/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Replicação Viral , Proteínas Quinases/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Grânulos Citoplasmáticos/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismoRESUMO
The nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, efficiently suppressed protein synthesis in mammalian cells. Unlike the nsp1 protein of severe acute respiratory syndrome coronavirus, a betacoronavirus, the TGEV nsp1 protein was unable to bind 40S ribosomal subunits or promote host mRNA degradation. TGEV nsp1 also suppressed protein translation in cell-free HeLa cell extract; however, it did not affect translation in rabbit reticulocyte lysate (RRL). Our data suggested that HeLa cell extracts and cultured host cells, but not RRL, contain a host factor(s) that is essential for TGEV nsp1-induced translational suppression.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas/efeitos dos fármacos , Vírus da Gastroenterite Transmissível/patogenicidade , Proteínas não Estruturais Virais/farmacologia , Animais , Extratos Celulares , Linhagem Celular , Células HeLa/virologia , Humanos , Rim/citologia , Rim/virologia , Masculino , Coelhos , Reticulócitos/virologia , Suínos , Testículo/citologia , Testículo/virologia , Proteínas não Estruturais Virais/metabolismoRESUMO
The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.