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1.
Arch Virol ; 169(7): 146, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38864914

RESUMO

Adenoviruses are a diverse group of viruses that can cause a variety of diseases in poultry, including respiratory and gastrointestinal infections. In turkeys (Meleagris gallopavo), adenoviruses commonly cause hemorrhagic enteritis and, rarely, inclusion body hepatitis. In this study, we investigated fowl adenoviruses (FAdVs) circulating in turkeys in Egypt. Following clinical examination of 500 birds, a portion of the hexon gene was amplified from four out of 50 samples from diseased birds (8%), and one amplicon that produced a strong band was selected for sequencing. Molecular and phylogenetic analysis revealed that the virus in that sample belonged to serotype FAdV-8b. Histopathological and immunohistochemical examinations of prepared tissue sections were performed to confirm the pathological findings. Diseased birds exhibited ruffled feathers, low body weight, a crouching posture, and diarrhea. Gross examination revealed petechial hemorrhage on the spleen, swollen pale liver, and congested intestine. Microscopic examination revealed the presence of eosinophilic and basophilic intranuclear inclusion bodies, nuclear pyknosis, and apoptotic bodies in the liver, congestion, hemorrhage, and fibrosis in the lungs, and desquamation of enterocytes. The presence of viral antigens in the liver, lungs, and intestine was confirmed by immunohistochemistry. To our knowledge, this is the first report of the characterization of an outbreak of inclusion body hepatitis in turkeys (hybrid converter breeds) due to FAdV-8b in Egypt. This finding raises an epidemiological alarm, necessitating further studies, including full-genome sequencing, to trace the virus's origin and genetic diversity.


Assuntos
Infecções por Adenoviridae , Aviadenovirus , Doenças das Aves Domésticas , Perus , Animais , Infecções por Adenoviridae/veterinária , Infecções por Adenoviridae/virologia , Infecções por Adenoviridae/patologia , Aviadenovirus/genética , Aviadenovirus/classificação , Aviadenovirus/isolamento & purificação , Proteínas do Capsídeo/genética , Egito , Hepatite Viral Animal/virologia , Hepatite Viral Animal/patologia , Corpos de Inclusão Viral/virologia , Fígado/virologia , Fígado/patologia , Filogenia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/patologia , Perus/virologia
2.
J Virol ; 96(18): e0090022, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36040180

RESUMO

Many negative-sense RNA viruses, including the highly pathogenic Ebola virus (EBOV), use cytoplasmic inclusion bodies (IBs) for viral RNA synthesis. However, it remains unclear how viral mRNAs are exported from these IBs for subsequent translation. We recently demonstrated that the nuclear RNA export factor 1 (NXF1) is involved in a late step in viral protein expression, i.e., downstream of viral mRNA transcription, and proposed it to be involved in this mRNA export process. We now provide further evidence for this function by showing that NXF1 is not required for translation of viral mRNAs, thus pinpointing its function to a step between mRNA transcription and translation. We further show that RNA binding of both NXF1 and EBOV NP is necessary for export of NXF1 from IBs, supporting a model in which NP hands viral mRNA over to NXF1 for export. Mapping of NP-NXF1 interactions allowed refinement of this model, revealing two separate interaction sites, one of them directly involving the RNA binding cleft of NP, even though these interactions are RNA-independent. Immunofluorescence analyses demonstrated that individual NXF1 domains are sufficient for its recruitment into IBs, and complementation assays helped to define NXF1 domains important for its function in the EBOV life cycle. Finally, we show that NXF1 is also required for protein expression of other viruses that replicate in cytoplasmic IBs, including Lloviu and Junín virus. These data suggest a role for NXF1 in viral mRNA export from IBs for various viruses, making it a potential target for broadly active antivirals. IMPORTANCE Filoviruses such as the Ebola virus (EBOV) cause severe hemorrhagic fevers with high case fatality rates and limited treatment options. The identification of virus-host cell interactions shared among several viruses would represent promising targets for the development of broadly active antivirals. In this study, we reveal the mechanistic details of how EBOV usurps the nuclear RNA export factor 1 (NXF1) to export viral mRNAs from viral inclusion bodies (IBs). We further show that NXF1 is not only required for the EBOV life cycle but also necessary for other viruses known to replicate in cytoplasmic IBs, including the filovirus Lloviu virus and the highly pathogenic arenavirus Junín virus. This suggests NXF1 as a promising target for the development of broadly active antivirals.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Proteínas de Transporte Nucleocitoplasmático , RNA Viral , Proteínas de Ligação a RNA , Antivirais , Ebolavirus/genética , Ebolavirus/metabolismo , Humanos , Corpos de Inclusão Viral/metabolismo , Corpos de Inclusão Viral/virologia , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Avian Dis ; 64(3): 330-334, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33205180

RESUMO

Fowl adenovirus (FAdV) type 8b isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2018 to 2019 were characterized serologically and genetically. Serologically, all isolates were well neutralized by antisera against the FAdV-8b strain, but they were not neutralized by antisera against the FAdV-8a strain. Phylogenetic analysis of the part of the hexon protein gene that includes the L1 region revealed that these isolates were all identical. They were also identical to foreign strains such as the SD1356 strain isolated in China and belonged to FAdV-8b. Furthermore, the 2018-19 Japanese IBH 8b isolates were genetically identical to the SD1356 strain by phylogenetic analysis of fiber genes, but they were different from previous Japanese 8b strains. These findings suggest that the 2018-19 Japanese IBH isolates might have been introduced from other countries.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/fisiologia , Galinhas , Hepatite Viral Animal/virologia , Corpos de Inclusão Viral/virologia , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/virologia , Animais , Japão , Filogenia
4.
Biochim Biophys Acta Mol Cell Res ; 1867(12): 118831, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32835749

RESUMO

Viruses reshape the organization of the cell interior to achieve different steps of their cellular cycle. Particularly, viral replication and assembly often take place in viral factories where specific viral and cellular proteins as well as nucleic acids concentrate. Viral factories can be either membrane-delimited or devoid of any cellular membranes. In the latter case, they are referred as membrane-less replication compartments. The most emblematic ones are the Negri bodies, which are inclusion bodies that constitute the hallmark of rabies virus infection. Interestingly, Negri bodies and several other viral replication compartments have been shown to arise from a liquid-liquid phase separation process and, thus, constitute a new class of liquid organelles. This is a paradigm shift in the field of virus replication. Here, we review the different aspects of membrane-less virus replication compartments with a focus on the Mononegavirales order and discuss their interactions with the host cell machineries and the cytoskeleton. We particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells.


Assuntos
Corpos de Inclusão Viral/genética , Raiva/genética , Compartimentos de Replicação Viral , Replicação Viral/genética , Membrana Celular/genética , Corpos de Inclusão Viral/virologia , Raiva/virologia , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Proteínas Virais/genética
5.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31776279

RESUMO

On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membraneless sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation, and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membraneless protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, relocalization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N terminus of the ATI or large truncations are made at the N or C termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation.IMPORTANCE Poxvirus genome replication, transcription, translation, and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.


Assuntos
Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/genética , Citoplasma/metabolismo , Replicação do DNA , Fator de Iniciação 4E em Eucariotos/metabolismo , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão Viral/virologia , Poxviridae/genética , Poxviridae/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Mensageiro/genética , Motivos de Ligação ao RNA/genética , Ribossomos/metabolismo , Vaccinia virus/genética , Proteínas Virais/genética , Vírion/metabolismo , Montagem de Vírus/genética
6.
J Virol ; 93(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31375591

RESUMO

Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells.IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally ∼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.


Assuntos
Corpos de Inclusão Viral/fisiologia , Vírus do Sarampo/fisiologia , Sarampo/virologia , Nucleoproteínas/metabolismo , Organelas/fisiologia , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Citoplasma/virologia , Células HeLa , Humanos , Corpos de Inclusão Viral/virologia , Extração Líquido-Líquido , Proteínas do Nucleocapsídeo , Nucleoproteínas/genética , Organelas/virologia , Fosfoproteínas/genética , Proteínas Virais/genética
7.
Virology ; 533: 68-76, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31125854

RESUMO

Reoviruses are thought to replicate and assemble in special cytoplasmic structures called 'viroplasms'. However, little is known about the viroplasms of the insect reoviruses, the cypoviruses. To investigate the viroplasm of Dendrolimus punctatus cypovirus (DpCPV), all proteins encoded by the 10 genomic segments of DpCPV were expressed in Sf9 cells using the Bac-to-Bac system. The viral nonstructural protein NSP2 formed viroplasm-like dots which showed close apposition with the endoplasmic reticulum and were surrounded by intracellular membranes during transfection. Colocalization and coimmunoprecipitation assays showed that NSP2 interacts with 4 of 6 structural proteins and another 2 nonstructural proteins, while NSP1 only colocalized with VP4, and NSP3 did not colocalize with any structural protein. Immunoelectron microscopy revealed that NSP2 were nearby the endoplasmic reticulum and mitochondria, and viral particles were present in the electron-dense inclusions formed by NSP2. We proposed that NSP2 is responsible for forming the viroplasms structures of DpCPV.


Assuntos
Corpos de Inclusão Viral/virologia , Reoviridae/metabolismo , Spodoptera/virologia , Proteínas não Estruturais Virais/metabolismo , Animais , Ligação Proteica , Reoviridae/genética , Células Sf9 , Proteínas não Estruturais Virais/genética
8.
J Avian Med Surg ; 33(1): 1-6, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31124605

RESUMO

Inclusion body hepatitis in falcons is caused by a herpesvirus designated Falconid HV-1. This herpesvirus and other herpesviruses affecting birds of prey have not been assigned to a genus and include inclusion body herpesvirus hepatitis in eagles (Accipitrid HV-1) and inclusion body herpesvirus hepatitis in owls (Strigid HV-1). Herpesvirus infections have been diagnosed in both captive and free-living raptors across Europe, North America, and Asia in different species of the family Falconidae. Herpesviruses affecting owls and falcons have been found to be antigenically similar to pigeon herpesvirus (Columbid HV-1) and distinct from other avian herpesviruses. When the herpesvirus isolates from owls, falcons, and pigeons were compared by sequencing a fragment of the herpes viral DNA polymerase gene from those birds naturally infected with the virus, the sequences from these 3 sources were found to be nearly identical. The authors of this study concluded that the Falconid HV-1, Strigid HV-1, and Columbid HV-1 were the same virus. Furthermore, the authors also proposed that the virus therefore be referred to as Columbid HV-1 (CoHV-1), because pigeons may be responsible for the transmission of the virus to birds of prey. Pigeons are often carriers of the virus without showing any clinical signs. It has long been suspected that raptors may contract the infection by the ingestion of infected pigeons. Some studies have suggested that falcons may not contract the infection through the oral route by ingesting carrier pigeons, but through the ocular or nasal route. Inclusion body herpesvirus hepatitis is a frequently diagnosed disease in the captive falcon population used for falconry, racing, and breeding in the Middle East, and it seems to be associated with the extensive use of pigeons for training and as a food item. This paper reviews the clinical and pathological findings in falcons affected by inclusion body herpesvirus hepatitis in the Middle East.


Assuntos
Doenças das Aves/patologia , Falconiformes , Hepatite Animal/patologia , Herpesviridae/classificação , Corpos de Inclusão Viral/virologia , Animais , Doenças das Aves/diagnóstico , Doenças das Aves/terapia , Doenças das Aves/virologia , Hepatite Animal/diagnóstico , Hepatite Animal/terapia , Hepatite Animal/virologia , Herpesviridae/isolamento & purificação , Oriente Médio
9.
PLoS Pathog ; 15(4): e1007733, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31034506

RESUMO

Formation of cytoplasmic inclusion bodies (IBs) is a hallmark of infections with non-segmented negative-strand RNA viruses (order Mononegavirales). We show here that Nipah virus (NiV), a bat-derived highly pathogenic member of the Paramyxoviridae family, differs from mononegaviruses of the Rhabdo-, Filo- and Pneumoviridae families by forming two types of IBs with distinct localizations, formation kinetics, and protein compositions. IBs in the perinuclear region form rapidly upon expression of the nucleocapsid proteins. These IBperi are highly mobile and associate with the aggresome marker y-tubulin. IBperi can recruit unrelated overexpressed cytosolic proteins but do not contain the viral matrix (M) protein. Additionally, NiV forms an as yet undescribed IB population at the plasma membrane (IBPM) that is y-tubulin-negative but contains the M protein. Infection studies with recombinant NiV revealed that IBPM require the M protein for their formation, and most likely represent sites of NiV assembly and budding. The identification of this novel type of plasma membrane-associated IBs not only provides new insights into NiV biology and may open new avenues to develop novel antiviral approaches to treat these highly pathogenic viruses, it also provides a basis for a more detailed characterization of IBs and their role in virus assembly and replication in infections with other Mononegavirales.


Assuntos
Membrana Celular/virologia , Infecções por Henipavirus/virologia , Corpos de Inclusão Viral/virologia , Vírus Nipah/patogenicidade , Proteínas da Matriz Viral/metabolismo , Animais , Chlorocebus aethiops , Glicoproteínas/metabolismo , Infecções por Henipavirus/metabolismo , Infecções por Henipavirus/patologia , Humanos , Corpos de Inclusão Viral/metabolismo , Corpos de Inclusão Viral/patologia , Células Vero , Montagem de Vírus , Internalização do Vírus
10.
Avian Pathol ; 48(3): 278-283, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30663340

RESUMO

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises. RESEARCH HIGHLIGHTS Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A. Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis. Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows. Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).


Assuntos
Aviadenovirus/classificação , Galliformes/virologia , Doenças das Aves Domésticas/diagnóstico , Traqueíte/veterinária , Animais , Aviadenovirus/genética , Aviadenovirus/isolamento & purificação , Brasil , Evolução Fatal , Adenovirus A das Aves/genética , Corpos de Inclusão Viral/virologia , Corpos de Inclusão Intranuclear/virologia , Necrose/veterinária , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Traqueia/patologia , Traqueia/virologia , Traqueíte/diagnóstico , Traqueíte/patologia , Traqueíte/virologia
11.
PLoS One ; 14(12): e0227004, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31891942

RESUMO

The present study was conducted in order to isolate, identify and characterize fowl aviadenovirus associated with inclusion body hepatitis (IBH) in three poultry farms (two of broiler chickens and one of breeder broiler chickens) in Morocco during 2015. Liver samples collected from affected three poultry farms were examined by histopathological examination. Tissue samples showing necrosis of hepatocytes associated with basophilic intranuclear inclusion bodies were homogenized and submitted to FAdV isolation in chicken embryo fibroblast (CEF) cell cultures and in SPF embryonated eggs. The cytopathic effect (CPE) was observed in the second passage with swelling and rounding of infected cells. The inoculated embryos were hemorrhagic and showed hepatitis with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The presence of the virus was confirmed by conventional polymerase chain reaction based on hexon gene from all investigated samples. Moreover, phylogenetic analysis of the hexon gene revealed that FAdVs isolated from different affected poultry belonged to FAdV 11 serotype of the D genotype group. This work is the first isolation in cell culture and SPF embryonated eggs of FAdV from Moroccan broilers and breeder broiler chickens with IBH.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/genética , Galinhas/virologia , Hepatite Viral Animal/virologia , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/classificação , Aviadenovirus/imunologia , Aviadenovirus/isolamento & purificação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Embrião de Galinha , DNA Viral/genética , DNA Viral/isolamento & purificação , Surtos de Doenças/veterinária , Hepatite Viral Animal/epidemiologia , Corpos de Inclusão Viral/virologia , Fígado/virologia , Marrocos/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Doenças das Aves Domésticas/epidemiologia , Sorogrupo , Sorotipagem
12.
J Virol ; 92(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30258011

RESUMO

Rotavirus replicates in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), the composition and structure of which have yet to be understood. Based on the analysis of a few proteins, earlier studies reported that rotavirus infection inhibits stress granule (SG) formation and disrupts P bodies (PBs). However, the recent demonstration that rotavirus infection induces cytoplasmic relocalization and colocalization with VMs of several nuclear hnRNPs and AU-rich element-binding proteins (ARE-BPs), which are known components of SGs and PBs, suggested the possibility of rotavirus-induced remodeling of SGs and PBs, prompting us to analyze a large number of the SG and PB components to understand the status of SGs and PBs in rotavirus-infected cells. Here we demonstrate that rotavirus infection induces molecular triage by selective exclusion of a few proteins of SGs (G3BP1 and ZBP1) and PBs (DDX6, EDC4, and Pan3) and sequestration of the remodeled/atypical cellular organelles, containing the majority of their components, in the VM. The punctate SG and PB structures are seen at about 4 h postinfection (hpi), coinciding with the appearance of small VMs, many of which fuse to form mature large VMs with progression of infection. By use of small interfering RNA (siRNA)-mediated knockdown and/or ectopic overexpression, the majority of the SG and PB components, except for ADAR1, were observed to inhibit viral protein expression and virus growth. In conclusion, this study demonstrates that VMs are highly complex supramolecular structures and that rotavirus employs a novel strategy of sequestration in the VM and harnessing of the remodeled cellular RNA recycling bins to promote its growth.IMPORTANCE Rotavirus is known to replicate in specialized virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), but the composition and structure of VMs are not yet understood. Here we demonstrate that rotavirus interferes with normal SG and PB assembly but promotes formation of atypical SG-PB structures by selective exclusion of a few components and employs a novel strategy of sequestration of the remodeled SG-PB granules in the VMs to promote virus growth by modulating their negative influence on virus infection. Rotavirus VMs appear to be complex supramolecular structures formed by the union of the triad of viral replication complexes and remodeled SGs and PBs, as well as other host factors, and designed to promote productive virus infection. These observations have implications for the planning of future research with the aim of understanding the structure of the VM, the mechanism of morphogenesis of the virus, and the detailed roles of host proteins in rotavirus biology.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Corpos de Inclusão Viral/virologia , Proteínas de Ligação a RNA/metabolismo , Rotavirus/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Corpos de Inclusão Viral/metabolismo , Infecções por Rotavirus/metabolismo , Infecções por Rotavirus/virologia , Replicação Viral
13.
mBio ; 9(4)2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087167

RESUMO

Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and µNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.IMPORTANCE Viruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus σNS and µNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.


Assuntos
Retículo Endoplasmático/metabolismo , Interações Hospedeiro-Patógeno , Corpos de Inclusão Viral/metabolismo , Reoviridae/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/virologia , Corpos de Inclusão Viral/virologia , Microscopia Intravital , Microscopia , Microscopia Eletrônica
14.
Avian Pathol ; 47(4): 384-390, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29587493

RESUMO

During the period from 2015 to 2017, frequent outbreaks of inclusion body hepatitis (IBH) were observed in broiler chickens and falcons in Saudi Arabia. Fifty samples were collected from both species. The histopathological examination and polymerase chain reaction confirmed the IBH infection in eight samples (five samples from chickens and three samples from falcons). The genomic sequence and phylogenetic analysis based on nucleotide and amino acid sequences of Saudi strains, reference fowl aviadenoviruses (FAdVs) and field viruses available in Genbank revealed that all investigated FAdVs clustered into FAdV-2 (species D) and FAdV-6 (species E). The host-dependent characterization revealed that falcon origin strains showed low identity (∼35%) with falcon adenoviruses isolated from USA, which clustered into a separate group. The identification of FAdV-D and FAdV-E in diseased falcons and chickens indicates cross-species transmission although falcons and chickens are phylogenetically different. The control of IBH infection in falcons and chickens should be based on the separation of carriers and susceptible chickens as well as falcons to prevent cross-species contact. Vaccination is an important method for prevention of IBH. The characterization of newly emerging FAdV strains provides valuable information for the development of an efficacious control strategy based on the molecular structure of current circulating FAdV strains in different species of birds.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/classificação , Doenças das Aves/transmissão , Galinhas/virologia , Surtos de Doenças/veterinária , Hepatite Viral Animal/transmissão , Corpos de Inclusão Viral/virologia , Adenoviridae/classificação , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/transmissão , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/genética , Aviadenovirus/isolamento & purificação , Doenças das Aves/epidemiologia , Doenças das Aves/virologia , Proteínas do Capsídeo/genética , Falconiformes , Hepatite Viral Animal/epidemiologia , Hepatite Viral Animal/virologia , Especificidade de Hospedeiro , Filogenia , Arábia Saudita/epidemiologia
15.
J Gen Virol ; 99(4): 585-595, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29465345

RESUMO

Autographa californica nucleopolyhedrovirus (AcMNPV) orf5 (ac5) is a group I alphabaculovirus-specific gene of unknown function, although the protein (AC5) was previously reported to be associated with the per os infectivity factor (PIF) complex. The purpose of this study was to study the dynamics of AC5 during AcMNPV infection and to verify whether it is indeed a component of the PIF complex. Transcription and expression analyses suggested that ac5 is a late viral gene. An ac5-deleted recombinant AcMNPV was generated by homologous recombination. A one-step growth curve assay indicated that ac5 was not required for budded virus (BV) production in Sf9 cells. Scanning electron microscopy and transmission electron microscopy demonstrated that the deletion of ac5 did not affect occlusion body (OB) morphology, and nor did it affect the insertion of occlusion-derived virus (ODV) into OBs. Partially denaturing SDS-PAGE and a co-immunoprecipitation assay clearly showed that AC5 was not a component of the PIF complex, while the deletion of ac5 did not affect the formation and presence of the PIF complex. Further analyses showed, however, that AC5 was an OB-specific protein, but it was not detected as a component of BVs or ODVs. Bioassay experiments showed that the oral infectivity of ac5-deleted AcMNPV to third instar Spodoptera exigua larvae was not significantly different from that of the ac5-repaired virus. In conclusion, AC5 is an intrinsic protein of OBs, instead of being a component of the PIF complex, and is not essential for either BV or ODV infection. AC5 is awaiting the assignment of another hitherto unknown function.


Assuntos
Corpos de Inclusão Viral/virologia , Nucleopoliedrovírus/metabolismo , Spodoptera/virologia , Proteínas Virais/metabolismo , Animais , Deleção de Genes , Larva/crescimento & desenvolvimento , Larva/virologia , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/crescimento & desenvolvimento , Células Sf9 , Spodoptera/crescimento & desenvolvimento , Proteínas Virais/genética
16.
PLoS Pathog ; 14(1): e1006769, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29309427

RESUMO

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Corpos de Inclusão Viral/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Química Click , Deleção de Genes , Herpes Simples/tratamento farmacológico , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Corpos de Inclusão Viral/efeitos dos fármacos , Corpos de Inclusão Viral/patologia , Corpos de Inclusão Viral/virologia , Cinética , Lisogenia/efeitos dos fármacos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína da Leucemia Promielocítica/antagonistas & inibidores , Proteína da Leucemia Promielocítica/genética , Interferência de RNA , Inibidores da Transcriptase Reversa/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteínas Virais/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
17.
J Biol Chem ; 292(50): 20379-20393, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29046350

RESUMO

Promyelocytic leukemia nuclear bodies (PML-NB) are sub-nuclear organelles that are the hub of numerous proteins. DNA/RNA viruses often hijack the cellular factors resident in PML-NBs to promote their proliferation in host cells. Hepatitis B virus (HBV), belonging to Hepadnaviridae family, remains undetected in early infection as it does not induce the innate immune response and is known to be the cause of several hepatic diseases leading to cirrhosis and hepatocellular carcinoma. The association of PML-NB proteins and HBV is being addressed in a number of recent studies. Here, we report that the PML-NB protein Speckled 110 kDa (Sp110) is SUMO1-modified and undergoes a deSUMOylation-driven release from the PML-NB in the presence of HBV. Intriguingly, Sp110 knockdown significantly reduced viral DNA load in the culture supernatant by activation of the type I interferon-response pathway. Furthermore, we found that Sp110 differentially regulates several direct target genes of hepatitis B virus protein X (HBx), a viral co-factor. Subsequently, we identified Sp110 as a novel interactor of HBx and found this association to be essential for the exit of Sp110 from the PML-NB during HBV infection and HBx recruitment on the promoter of these genes. HBx, in turn, modulates the recruitment of its associated transcription cofactors p300/HDAC1 to these co-regulated genes, thereby altering the host gene expression program in favor of viral persistence. Thus, we report a mechanism by which HBV can evade host immune response by hijacking the PML-NB protein Sp110, and therefore, we propose it to be a novel target for antiviral therapy.


Assuntos
Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatócitos/metabolismo , Corpos de Inclusão Viral/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/metabolismo , Sumoilação , Transativadores/fisiologia , Apoptose , Biomarcadores/sangue , Biomarcadores/metabolismo , DNA Viral/metabolismo , Regulação Bacteriana da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/patologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Corpos de Inclusão Viral/patologia , Corpos de Inclusão Viral/virologia , Antígenos de Histocompatibilidade Menor/sangue , Antígenos de Histocompatibilidade Menor/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/sangue , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transporte Proteico , Interferência de RNA , Carga Viral , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias , Fenômenos Fisiológicos Virais , Replicação Viral
18.
Appl Environ Microbiol ; 83(14)2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28500037

RESUMO

The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.


Assuntos
Corpos de Inclusão Viral/virologia , Nucleopoliedrovírus/fisiologia , Nucleopoliedrovírus/patogenicidade , Animais , Expressão Gênica , Corpos de Inclusão Viral/genética , Corpos de Inclusão Viral/metabolismo , Larva/genética , Larva/metabolismo , Larva/virologia , Nucleopoliedrovírus/genética , Proteínas de Matriz de Corpos de Inclusão , Regiões Promotoras Genéticas , Spodoptera/genética , Spodoptera/metabolismo , Spodoptera/virologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Virulência
19.
PLoS Pathog ; 12(10): e1005942, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27749929

RESUMO

Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Corpos de Inclusão Viral/virologia , Vírus da Raiva , Raiva/virologia , Replicação Viral/fisiologia , Animais , Western Blotting , Linhagem Celular , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Hibridização in Situ Fluorescente , Microscopia Confocal , Raiva/imunologia , Vírus da Raiva/imunologia , Reação em Cadeia da Polimerase em Tempo Real
20.
J Gen Virol ; 97(11): 2926-2938, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27580912

RESUMO

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.


Assuntos
Carcinoma de Célula de Merkel/metabolismo , Corpos de Inclusão Viral/metabolismo , Poliomavírus das Células de Merkel/fisiologia , Infecções por Polyomavirus/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Infecções Tumorais por Vírus/metabolismo , Replicação Viral , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/virologia , Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Humanos , Corpos de Inclusão Viral/genética , Corpos de Inclusão Viral/virologia , Poliomavírus das Células de Merkel/genética , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia , Proteína da Leucemia Promielocítica/genética , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia
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