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1.
Int J Mol Sci ; 22(24)2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34948288

RESUMO

The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses.


Assuntos
Vírus de RNA de Cadeia Dupla/genética , Genoma Viral/genética , Vírus Auxiliares/genética , RNA de Cadeia Dupla/genética , Torulaspora/genética , Vinho/microbiologia , Vinho/virologia , Sequência de Aminoácidos , Sequência de Bases , Capsídeo , RNA Viral/genética , Saccharomyces cerevisiae/genética
2.
PLoS Pathog ; 17(6): e1009638, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34061891

RESUMO

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 Parvoviridae
3.
Sci Rep ; 11(1): 10400, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34002008

RESUMO

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0-3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.


Assuntos
Conectoma/métodos , Hipotálamo/diagnóstico por imagem , Imagem Molecular/métodos , Neurônios/metabolismo , Receptores para Leptina/análise , Animais , Proteínas Aviárias/genética , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vírus Auxiliares/genética , Hipotálamo/citologia , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Vírus da Raiva/genética , Receptores para Leptina/metabolismo , Receptores Virais/genética , Núcleos Septais/citologia , Núcleos Septais/diagnóstico por imagem , Núcleos Septais/metabolismo , Técnicas Estereotáxicas
4.
J Virol ; 95(13): e0048621, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33853961

RESUMO

Wild-type adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by coinfecting helper viruses such as adenoviruses and herpesviruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high-throughput sequencing, we found that herpes simplex virus 1 (HSV-1) coinfection leads to a shift in the type of AAV genome end recombination. In particular, open-end inverted terminal repeat (ITR) recombination was enhanced, whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1-mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 coinfection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified. IMPORTANCE Adeno-associated virus (AAV)-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical-grade vector, including methods that employ helper factors derived from herpes simplex virus 1 (HSV-1). Yet, to date, we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.


Assuntos
Dependovirus/crescimento & desenvolvimento , Dependovirus/genética , Genoma Viral/genética , Vírus Auxiliares/genética , Herpesvirus Humano 1/genética , Recombinação Genética/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Coinfecção/patologia , Células HEK293 , Células HeLa , Herpes Simples/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infecções por Parvoviridae/patologia , Sequências Repetidas Terminais/genética , Células Vero , Interferência Viral/genética , Replicação Viral/genética
5.
J Mol Biol ; 433(9): 166896, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33639215

RESUMO

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , DNA Recombinante/genética , Edição de Genes/métodos , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Epitopos/genética , Epitopos/imunologia , Genes Virais/genética , Marcadores Genéticos/genética , Vetores Genéticos/genética , Genoma Viral/genética , Vírus Auxiliares/genética , Fusão de Membrana , Vírion/genética , Internalização do Vírus
6.
Virus Genes ; 57(1): 1-22, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33226576

RESUMO

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 Viral
7.
Viruses ; 12(9)2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867300

RESUMO

P4 is a mobile genetic element (MGE) that can exist as a plasmid or integrated into its Escherichia coli host genome, but becomes packaged into phage particles by a helper bacteriophage, such as P2. P4 is the original example of what we have termed "molecular piracy", the process by which one MGE usurps the life cycle of another for its own propagation. The P2 helper provides most of the structural gene products for assembly of the P4 virion. However, when P4 is mobilized by P2, the resulting capsids are smaller than those normally formed by P2 alone. The P4-encoded protein responsible for this size change is called Sid, which forms an external scaffolding cage around the P4 procapsids. We have determined the high-resolution structure of P4 procapsids, allowing us to build an atomic model for Sid as well as the gpN capsid protein. Sixty copies of Sid form an intertwined dodecahedral cage around the T = 4 procapsid, making contact with only one out of the four symmetrically non-equivalent copies of gpN. Our structure provides a basis for understanding the sir mutants in gpN that prevent small capsid formation, as well as the nms "super-sid" mutations that counteract the effect of the sir mutations, and suggests a model for capsid size redirection by Sid.


Assuntos
Bacteriófagos/química , Proteínas do Capsídeo/química , Capsídeo/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Vírus Auxiliares/química , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo , Mutação , Conformação Proteica , Vírus Satélites/química , Vírus Satélites/genética , Vírus Satélites/metabolismo
8.
BMB Rep ; 53(11): 565-575, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32958121

RESUMO

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replicationcompetent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases. [BMB Reports 2020; 53(11): 565-575].


Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Terapia Genética/métodos , Linhagem Celular , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo , Humanos , Integrases/genética , Plasmídeos/genética , Proteínas Virais/genética
9.
Virus Res ; 286: 198075, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32592818

RESUMO

Reverse genetics technology allows one to engineer replication-competent viruses from cloned cDNAs at will. Since the establishment of the initial reverse genetics system for species A rotaviruses (RVAs) requiring a helper virus in 2006, attempts have been successfully made to improve this technology. Efficient generation of replication-competent RVAs is now possible from just 11 T7-driven plasmids encoding an RVA genome when the quantity ratio of the two rescue T7-driven plasmids for the NSP2 and NSP5 segments is increased by 3-fold in relation to that of the other nine plasmids (11 plasmid-only system). Further, it is now possible to generate recombinant RVAs even with severely less efficient infectivity by using the 11 plasmid-only system, which has not been possible with the existing approaches. More importantly, the 11 plasmid-only system does not need any helper expression plasmid, and thus this simplest and robust system has a clear advantage over the existing systems in terms of safety. This 11 plasmid-only system should contribute to the development of safe next-generation vaccines and vaccine vectors.


Assuntos
Genoma Viral , Genética Reversa , Rotavirus/genética , Animais , Linhagem Celular , DNA Complementar/genética , Vírus Auxiliares/genética , Humanos , Camundongos , RNA Viral/genética , Proteínas não Estruturais Virais/genética , Replicação Viral
10.
Viruses ; 12(6)2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32575422

RESUMO

The adeno-associated virus (AAV) is a small, nonpathogenic parvovirus, which depends on helper factors to replicate. Those helper factors can be provided by coinfecting helper viruses such as adenoviruses, herpesviruses, or papillomaviruses. We review the basic biology of AAV and its most-studied helper viruses, adenovirus type 5 (AdV5) and herpes simplex virus type 1 (HSV-1). We further outline the direct and indirect interactions of AAV with those and additional helper viruses.


Assuntos
Adenoviridae/metabolismo , Dependovirus/crescimento & desenvolvimento , Vírus Auxiliares/metabolismo , Herpesvirus Humano 1/metabolismo , Replicação Viral/genética , Coinfecção/virologia , Dependovirus/genética , Humanos , Infecções por Parvoviridae/virologia , Proteínas Virais/genética
11.
Hum Gene Ther ; 31(9-10): 499-511, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303138

RESUMO

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 , Humanos
13.
mBio ; 11(2)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184255

RESUMO

Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particles. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.


Assuntos
Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/patogenicidade , Serpentes/virologia , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular Tumoral , Coinfecção/virologia , Genoma Viral , Vírus Auxiliares/genética , Vírus da Hepatite B/genética , Vírus Delta da Hepatite/classificação , Humanos , RNA Viral/genética , Tropismo Viral , Replicação Viral
14.
Hum Gene Ther ; 31(9-10): 553-564, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024384

RESUMO

Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.


Assuntos
Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Biodiversidade , Linhagem Celular , Evolução Molecular Direcionada , Biblioteca Gênica , Técnicas de Transferência de Genes , Genoma Viral , Células HaCaT , Vírus Auxiliares , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reprodutibilidade dos Testes , Transdução Genética , Replicação Viral
15.
Mol Brain ; 13(1): 5, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931837

RESUMO

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 Viral
16.
FEBS Lett ; 593(24): 3623-3648, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31705806

RESUMO

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 , Camundongos
17.
Hum Gene Ther ; 30(11): 1371-1384, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31456426

RESUMO

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 , Ratos
18.
RNA ; 25(11): 1432-1438, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31434792

RESUMO

R2 is a long interspersed element (LINE) found in a specific sequence of the 28S rDNA among a wide variety of animals. Recently, we observed that R2Ol isolated from medaka fish, Oryzias latipes, retrotransposes sequence specifically into the target sequence of zebrafish. Because the 28S target and flanking regions are widely conserved among vertebrates, we examined whether R2Ol can also integrate in a sequence-specific manner in human cells. Using adenovirus-mediated expression of R2Ol constructs, we confirmed an accurate insertion of R2Ol into the 28S target of human 293T cells. However, the R2Ol mutant devoid of endonuclease (EN) activity showed no retrotransposition ability, suggesting that the sequence-specific integration of R2Ol into 28S rDNA occurs via the cleavage activity of EN. By introducing both R2Ol helper virus and donor plasmid in human cells, we succeeded in retrotransposing an exogenous EGFP gene into the 28S target site by the trans-complementation system, which enabled simplification of specific gene knock-in in a time-efficient manner. We believe that R2Ol may provide an alternative targeted gene knock-in method for practical applications such as gene therapy in future.


Assuntos
DNA Ribossômico/genética , Elementos Nucleotídeos Longos e Dispersos , RNA Ribossômico 28S/genética , Retroelementos , Adenoviridae/genética , Técnicas de Introdução de Genes , Teste de Complementação Genética , Células HEK293 , Vírus Auxiliares/genética , Humanos , Plasmídeos
19.
Nucleic Acids Res ; 47(15): 8255-8271, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31269212

RESUMO

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 , Doenças das Plantas/virologia , RNA Viral/metabolismo , Tabaco/virologia , 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/genética
20.
J Gene Med ; 21(6): e3094, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31037799

RESUMO

BACKGROUND: Adeno-associated virus-based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. METHODS: In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome-containing particles using a real-time polymerase chain reaction and functional units were investigated by transduction assays. RESULTS: Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication-deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild-type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. CONCLUSIONS: The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild-type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.


Assuntos
Adenoviridae/genética , Dependovirus/genética , Engenharia Genética , Vetores Genéticos/genética , Mutação , Linhagem Celular , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vírus Auxiliares/genética , Humanos , Transdução Genética , Transfecção , Transgenes , Replicação Viral
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