RESUMO
Previous studies reported that the hepatitis C virus (HCV) could help disseminate the hepatitis D virus (HDV) in vivo through the unrelated hepatitis B virus (HBV), but with essentially inconclusive results. To try to shed light on this still-debated topic, 146 anti-HCV-positive subjects (of whom 91 HCV/HIV co-infected, and 43 with prior HCV eradication) were screened for anti-HDV antibodies (anti-HD), after careful selection for negativity to any serologic or virologic marker of current or past HBV infection. One single HCV/HIV co-infected patient (0.7%) tested highly positive for anti-HD, but with no positive HDV-RNA. Her husband, in turn, was a HCV/HIV co-infected subject with a previous contact with HBV. While conducting a thorough review of the relevant literature, the authors attempted to exhaustively describe the medical history of both the anti-HD-positive patient and her partner, believing it to be the key to dissecting the possible complex mechanisms of HDV transmission from one subject to another, and speculating that in the present case, it may have been HCV itself that behaved as an HDV helper virus. In conclusion, this preliminary research, while needing further validation in large prospective studies, provided some further evidence of a role of HCV in HDV dissemination in humans.
Assuntos
Coinfecção , Hepacivirus , Hepatite C , Hepatite D , Vírus Delta da Hepatite , Humanos , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/fisiologia , Hepacivirus/genética , Hepacivirus/fisiologia , Feminino , Hepatite C/virologia , Coinfecção/virologia , Masculino , Vírus Auxiliares/fisiologia , Anticorpos Anti-Hepatite/sangue , Adulto , Pessoa de Meia-Idade , Infecções por HIV/virologia , Infecções por HIV/complicações , RNA Viral , Hepatite B/virologiaRESUMO
As a type of parasitic agent, satellite RNAs (satRNAs) rely on cognate helper viruses to achieve their replication and transmission. During the infection of satRNAs, helper virus RNAs serve as templates for synthesizing viral proteins, including the replication proteins essential for satRNA replication. However, the role of non-template functions of helper virus RNAs in satRNA replication remains unexploited. Here we employed the well-studied model that is composed of cucumber mosaic virus (CMV) and its associated satRNA. In the experiments employing the CMV trans-replication system, we observed an unexpected phenomenon the replication proteins of the mild strain LS-CMV exhibited defective in supporting satRNA replication, unlike those of the severe strain Fny-CMV. Independent of translation products, all CMV genomic RNAs could enhance satRNA replication, when combined with the replication proteins of CMV. This enhancement is contingent upon the recruitment and complete replication of helper virus RNAs. Using the method developed for analyzing the satRNA recruitment, we observed a markedly distinct ability of the replication proteins from both CMV strains to recruit the positive-sense satRNA-harboring RNA3 mutant for replication. This is in agreement with the differential ability of both 1a proteins in binding satRNAs in plants. The discrepancies provide a convincing explanation for the variation of the replication proteins of both CMV strains in replicating satRNAs. Taken together, our work provides compelling evidence that the non-template functions of helper virus RNAs create an optimal replication environment to enhance satRNA proliferation.
Assuntos
Cucumovirus , Vírus Auxiliares , RNA Satélite , RNA Viral , Replicação Viral , Vírus Auxiliares/genética , Vírus Auxiliares/fisiologia , Cucumovirus/genética , Cucumovirus/metabolismo , Cucumovirus/fisiologia , RNA Satélite/metabolismo , RNA Satélite/genética , RNA Viral/genética , RNA Viral/metabolismo , Doenças das Plantas/virologia , Nicotiana/virologia , Nicotiana/metabolismo , Nicotiana/genética , Proteínas Virais/metabolismo , Proteínas Virais/genéticaRESUMO
Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.
Assuntos
Beta vulgaris , Doenças das Plantas , Vírus de Plantas , Vírus Satélites , Beta vulgaris/virologia , Doenças das Plantas/virologia , Vírus Satélites/genética , Vírus Satélites/fisiologia , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Vírus Auxiliares/genética , Vírus Auxiliares/fisiologia , RNA Viral/genética , Raízes de Plantas/virologia , Genoma Viral/genética , Microbiologia do SoloRESUMO
The genetic diversity of baculoviruses provides a sustainable agronomic solution when resistance to biopesticides seems to be on the rise. This genetic diversity promotes insect infection by several genotypes (i.e., multiple infections) that are more likely to kill the host. However, the mechanism and regulation of these virus interactions are still poorly understood. In this article, we focused on baculoviruses infecting the codling moth, Cydia pomonella: two Cydia pomonella granulovirus genotypes, CpGV-M and CpGV-R5, and Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). The influence of the order of ingestion of the virus genotypes, the existence of an ingestion delay between the genotypes and the specificity of each genotype involved in the success of multiple infection were studied in the case of Cydia pomonella resistance. To obtain a multiple infection in resistant insects, the order of ingestion is a key factor, but the delay for ingestion of the second virus is not. CrpeNPV cannot substitute CpGV-R5 to allow replication of CpGV-M.
Assuntos
Comportamento Alimentar , Granulovirus/genética , Granulovirus/fisiologia , Vírus Auxiliares/fisiologia , Mariposas/virologia , Replicação Viral , Animais , Variação Genética , Vírus Auxiliares/genéticaRESUMO
Hepatitis delta virus (HDV) is a defective human virus that lacks the ability to produce its own envelope proteins and is thus dependent on the presence of a helper virus, which provides its surface proteins to produce infectious particles. Hepatitis B virus (HBV) was so far thought to be the only helper virus described to be associated with HDV. However, recent studies showed that divergent HDV-like viruses could be detected in fishes, birds, amphibians, and invertebrates, without evidence of any HBV-like agent supporting infection. Another recent study demonstrated that HDV can be transmitted and propagated in experimental infections ex vivo and in vivo by different enveloped viruses unrelated to HBV, including hepatitis C virus (HCV) and flaviviruses such as Dengue and West Nile virus. All this new evidence, in addition to the identification of novel virus species within a large range of hosts in absence of HBV, suggests that deltaviruses may take advantage of a large spectrum of helper viruses and raises questions about HDV origins and evolution.
Assuntos
Vírus Auxiliares , Hepatite D/virologia , Vírus Delta da Hepatite , Animais , Evolução Molecular , Genoma Viral , Vírus Auxiliares/fisiologia , Vírus Delta da Hepatite/classificação , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/fisiologia , Especificidade de Hospedeiro , Humanos , Filogenia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3' inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.
Assuntos
Coinfecção , Dependovirus , Simplexvirus , Replicação Viral , Animais , Linhagem Celular , Genoma Viral , Vírus Auxiliares/fisiologia , Herpes Simples , Humanos , Infecções por ParvoviridaeRESUMO
The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids in vitro and suppresses tumor growth in two orthotopic lung cancer models in vivo. Treatment with CAd MSCs increases the overall numbers of human T cells in vivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.
Assuntos
Anticorpos Monoclonais/genética , Dependovirus/fisiologia , Vírus Auxiliares/fisiologia , Interleucina-12/metabolismo , Neoplasias Pulmonares/terapia , Células-Tronco Mesenquimais/virologia , Receptores de Antígenos de Linfócitos T/metabolismo , Células A549 , Animais , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular Tumoral , Terapia Combinada , Dependovirus/genética , Vírus Auxiliares/genética , Humanos , Imunoterapia Adotiva , Interleucina-12/antagonistas & inibidores , Interleucina-12/genética , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Terapia Viral Oncolítica , Receptor ErbB-2/imunologia , Microambiente Tumoral , Tropismo Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Plant viral satellites fall under the category of subviral agents. Their genomes are composed of small RNA or DNA molecules a few hundred nucleotides in length and contain an assortment of highly complex and overlapping functions. Each lacks the ability to either replicate or undergo encapsidation or both in the absence of a helper virus (HV). As the number of known satellites increases steadily, our knowledge regarding their sequence conservation strategies, means of replication and specific interactions with host and helper viruses is improving. This review demonstrates that the molecular interactions of these satellites are unique and highly complex, largely influenced by the highly specific host plants and helper viruses that they associate with. Circularized forms of single-stranded RNA are of particular interest, as they have recently been found to play a variety of novel cellular functions. Linear forms of satRNA are also of great significance as they may complement the helper virus genome in exacerbating symptoms, or in certain instances, actively compete against it, thus reducing symptom severity. This review serves to describe the current literature with respect to these molecular mechanisms in detail as well as to discuss recent insights into this emerging field in terms of evolution, classification and symptom development. The review concludes with a discussion of future steps in plant viral satellite research and development.
Assuntos
Doenças das Plantas/virologia , Vírus de Plantas , Vírus Satélites , DNA Satélite , DNA Viral , Vírus Auxiliares/fisiologia , Interações entre Hospedeiro e Microrganismos , Vírus de Plantas/genética , Vírus de Plantas/patogenicidade , Vírus de Plantas/fisiologia , RNA Satélite , RNA Viral , Vírus Satélites/genética , Vírus Satélites/patogenicidade , Vírus Satélites/fisiologia , Replicação ViralAssuntos
Vírus Auxiliares/fisiologia , Orthopoxvirus/genética , Biologia Sintética/legislação & jurisprudência , Animais , Doenças Transmissíveis Emergentes/prevenção & controle , Doenças Transmissíveis Emergentes/virologia , Doenças dos Cavalos/virologia , Cavalos , Disseminação de Informação , Infecções por Poxviridae/veterinária , Biologia Sintética/éticaRESUMO
Recombinant adeno-associated virus has emerged as one of the most promising gene therapy delivery vectors. Development of these vectors took advantage of key features of the wild-type adeno-associated virus (AAV), enabled by basic studies of the underlying biology and requirements for transcription, replication, and packaging of the viral genome. Each step in generating and utilizing viral vectors involves numerous molecular interactions that together determine the efficiency of vector production and gene delivery. Once delivered into the cell, interactions with host proteins will determine the fate of the viral genome, and these will impact the intended goal of gene delivery. Here, we provide an overview of known interactions of the AAV genome with viral and cellular proteins involved in its amplification, packaging, and expression. Further appreciation of how the AAV genome interacts with host factors will enhance how this simple virus can be harnessed for an array of vector purposes that benefit human health.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Interações entre Hospedeiro e Microrganismos , Transdução Genética , Replicação Viral , Animais , Técnicas de Transferência de Genes , Genoma Viral , Vírus Auxiliares/fisiologia , HumanosRESUMO
Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.
Assuntos
Transporte Axonal/fisiologia , Encéfalo/citologia , Vírus Defeituosos/fisiologia , Dependovirus/fisiologia , Proteínas de Fluorescência Verde/análise , Vírus Auxiliares/fisiologia , Herpesvirus Humano 1/fisiologia , Vias Neurais/ultraestrutura , Técnicas de Rastreamento Neuroanatômico/métodos , Marcadores do Trato Nervoso/análise , Neurônios/ultraestrutura , Vírus Reordenados/fisiologia , Animais , Linhagem Celular , Núcleo Celular/virologia , Vírus Defeituosos/genética , Dependovirus/genética , Genes Reporter , Genes Sintéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Vírus Auxiliares/genética , Herpesvirus Humano 1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/virologia , Vírus Reordenados/genética , Proteínas do Complexo da Replicase Viral/genética , Replicação ViralRESUMO
Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by ex vivo and in vivo editing in transgenic mice, nonhuman primates, as well as in human CD34+ cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.
Assuntos
Adenoviridae/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Vírus Auxiliares/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/química , Humanos , CamundongosRESUMO
The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.
Assuntos
Adenoviridae/fisiologia , Vírus Auxiliares/fisiologia , Inflamação/patologia , Inflamação/virologia , Retina/patologia , Retina/virologia , Transdução Genética , Animais , Morte Celular , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Células Fotorreceptoras de Vertebrados/patologia , RatosRESUMO
As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.
Assuntos
Satélite do Vírus do Mosaico do Pepino/metabolismo , Cucumovirus/fisiologia , Vírus Auxiliares/fisiologia , Nicotiana/virologia , Doenças das Plantas/virologia , RNA Viral/metabolismo , Sequência de Bases , Satélite do Vírus do Mosaico do Pepino/química , Satélite do Vírus do Mosaico do Pepino/genética , Cucumovirus/genética , Vírus Auxiliares/genética , Mutação , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Replicação Viral/genéticaRESUMO
Hepatitis delta virus, the only member of the only species in the genus Deltavirus, is a unique human pathogen. Its ~1.7 kb circular negative-sense RNA genome encodes a protein, hepatitis delta antigen, which occurs in two forms, small and large, both with unique functions. Hepatitis delta virus uses host RNA polymerase II to replicate via double rolling circle RNA synthesis. Newly synthesized linear RNAs are circularized after autocatalytic cleavage and ligation. Hepatitis delta virus requires the envelope of the helper virus, hepatitis B virus (family Hepadnaviridae), to produce infectious particles. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Deltavirus which is available at www.ictv.global/report/deltavirus.
Assuntos
Hepatite D/virologia , Vírus Delta da Hepatite/classificação , Vírus Delta da Hepatite/genética , RNA Viral/genética , Genoma Viral , Vírus Auxiliares/fisiologia , Vírus da Hepatite B/fisiologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Humanos , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Circular , RNA Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação ViralRESUMO
The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.
Assuntos
Micovírus/isolamento & purificação , Vírus Auxiliares/isolamento & purificação , Saccharomyces/virologia , Vírus Satélites/isolamento & purificação , Totiviridae/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micovírus/classificação , Micovírus/genética , Micovírus/fisiologia , Genoma Viral , Vírus Auxiliares/classificação , Vírus Auxiliares/genética , Vírus Auxiliares/fisiologia , Filogenia , Saccharomyces/genética , Saccharomyces/metabolismo , Vírus Satélites/classificação , Vírus Satélites/genética , Vírus Satélites/fisiologia , Totiviridae/classificação , Totiviridae/genética , Totiviridae/fisiologiaRESUMO
Panicum mosaic virus (PMV) is a helper RNA virus for satellite RNAs (satRNAs) and a satellite virus (SPMV). Here, we describe modifications that occur at the 3'-end of a satRNA of PMV, satS. Co-infections of PMV+satS result in attenuation of the disease symptoms induced by PMV alone in Brachypodium distachyon and proso millet. The 375 nt satS acquires ~100-200 nts from the 3'-end of PMV during infection and is associated with decreased abundance of the PMV RNA and capsid protein in millet. PMV-satS chimera RNAs were isolated from native infections of St. Augustinegrass and switchgrass. Phylogenetic analyses revealed that the chimeric RNAs clustered according to the host species from which they were isolated. Additionally, the chimera satRNAs acquired non-viral "linker" sequences in a host-specific manner. These results highlight the dynamic regulation of viral pathogenicity by satellites, and the selective host-dependent, sequence-based pressures for driving satRNA generation and genome compositions.
Assuntos
Vírus Auxiliares , Especificidade de Hospedeiro , Doenças das Plantas , RNA Satélite , Vírus Satélites , Tombusviridae , Brachypodium/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Genoma Viral , Vírus Auxiliares/genética , Vírus Auxiliares/fisiologia , Panicum/virologia , Filogenia , Doenças das Plantas/virologia , Poaceae/virologia , Recombinação Genética , RNA Satélite/genética , RNA Satélite/metabolismo , Vírus Satélites/genética , Vírus Satélites/fisiologia , Tombusviridae/genética , Tombusviridae/fisiologiaRESUMO
An attenuated rabies virus that expresses fluorescent protein has made it possible to analyze retrograde (presynaptic) monosynaptic connections in vivo. By combining attenuated rabies virus with a Cre-loxP based system to target cells in a subtype-specific fashion, it is possible to examine neuronal input in vivo onto any class of neuron, in development and in the mature brain. We describe here the methods to amplify deletion mutant, pseudotyped rabies virus, selectively target cells of interest using genetic and viral approaches, as well as the stereotaxic procedures required to target neuronal subtypes of interest in vivo.
Assuntos
Conectoma/métodos , Neurônios/fisiologia , Neurônios/virologia , Vírus da Raiva/fisiologia , Sinapses/fisiologia , Animais , Transporte Biológico , Linhagem Celular , Expressão Gênica , Genes Reporter , Vetores Genéticos , Vírus Auxiliares/fisiologia , Camundongos , Microscopia de Fluorescência , Sinapses/virologia , Tropismo Viral , Replicação ViralRESUMO
Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability.
Assuntos
Anticorpos/metabolismo , Bacteriófagos/fisiologia , Proteínas do Capsídeo/genética , Escherichia coli/virologia , Especificidade de Anticorpos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo , Vírus Auxiliares/fisiologia , Biblioteca de Peptídeos , Sinais Direcionadores de Proteínas , Vírion/genética , Vírion/metabolismo , Vírion/fisiologiaRESUMO
Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.