Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.442
Filtrar
1.
J Gen Virol ; 105(2)2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38421275

RESUMO

Kolmioviridae is a family for negative-sense RNA viruses with circular, viroid-like genomes of about 1.5-1.7 kb that are maintained in mammals, amphibians, birds, fish, insects and reptiles. Deltaviruses, for instance, can cause severe hepatitis in humans. Kolmiovirids encode delta antigen (DAg) and replicate using host-cell DNA-directed RNA polymerase II and ribozymes encoded in their genome and antigenome. They require evolutionary unrelated helper viruses to provide envelopes and incorporate helper virus proteins for infectious particle formation. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Kolmioviridae, which is available at ictv.global/report/kolmioviridae.


Assuntos
Vírus Auxiliares , Viroides , Animais , Humanos , Evolução Biológica , Vírus de RNA de Sentido Negativo , RNA Polimerase II , Mamíferos
2.
ISME J ; 17(12): 2381-2388, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37907733

RESUMO

Satellites are mobile genetic elements that are dependent upon the replication machinery of their helper viruses. Bacteriophages have provided many examples of satellite nucleic acids that utilize their helper morphogenic genes for propagation. Here we describe two novel satellite-helper phage systems, Mulch and Flayer, that infect Streptomyces species. The satellites in these systems encode for encapsidation machinery but have an absence of key replication genes, thus providing the first example of bacteriophage satellite viruses. We also show that codon usage of the satellites matches the tRNA gene content of the helpers. The satellite in one of these systems, Flayer, does not appear to integrate into the host genome, which represents the first example of a virulent satellite phage. The Flayer satellite has a unique tail adaptation that allows it to attach to its helper for simultaneous co-infection. These findings demonstrate an ever-increasing array of satellite strategies for genetic dependence on their helpers in the evolutionary arms race between satellite and helper phages.


Assuntos
Bacteriófagos , Streptomyces , Vírus Satélites/genética , Streptomyces/genética , Virulência , Vírus Auxiliares/genética , Bacteriófagos/genética
3.
Virol J ; 20(1): 228, 2023 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-37817259

RESUMO

Adeno-associated virus (AAV) differs from most other viruses, as it requires the simultaneous presence of a helper virus for an active infection. Up to 80% of the human population is seropositive for AAV antibodies. AAV has been known to be a non-pathogenic virus and an inhibitor of carcinogenesis caused by coinfecting viruses. However, the recent reports associating AAV infection with hepatocellular carcinoma development and the mysterious cases of acute severe hepatitis in children have challenged the idea that AAV is a harmless virus. Herein, we explore the usefulness of AAV in gene therapy and the importance of AAV as a protector or perpetrator in human carcinogenesis, ultimately reflecting on the dual role of AAV in human health.


Assuntos
Dependovirus , Neoplasias Hepáticas , Criança , Humanos , Dependovirus/genética , Replicação Viral , Vírus Auxiliares/genética , Carcinogênese
4.
J Gen Virol ; 104(6)2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37326617

RESUMO

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasatellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans-replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumulation of its helper virus DNA. For the first time, we have identified that the ToLCPaB-encoded ßC1 protein is able to interact with ToLCGV-encoded replication initiator protein (Rep). In addition, we demonstrate that the C-terminal region of ßC1 interacts with the C-terminus of Rep (RepC) protein. Our previous study had established that ßC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of ßC1 (ßC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity-deficient K49A mutated ßC1 (ßC1K49A) and RepC proteins revealed that Rep-ßC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we demonstrate that ßC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that the ßC1-interacting region of Rep protein encompasses its Walker-B and B' motifs. The results of docking studies supported that the ßC1-interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-ßC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest that ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.


Assuntos
Begomovirus , Geminiviridae , Geminiviridae/genética , Vírus Auxiliares , Lisina/metabolismo , Hidrólise , Proteínas Virais/genética , Proteínas Virais/metabolismo , Begomovirus/genética , Trifosfato de Adenosina/metabolismo , Doenças das Plantas
5.
Nature ; 617(7961): 574-580, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36996871

RESUMO

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.


Assuntos
Infecções por Adenovirus Humanos , Coinfecção , Dependovirus , Hepatite , Criança , Humanos , Doença Aguda , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , Coinfecção/epidemiologia , Coinfecção/virologia , Dependovirus/genética , Dependovirus/isolamento & purificação , Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/virologia , Hepatite/epidemiologia , Hepatite/virologia , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 6/isolamento & purificação , Enterovirus Humano A/isolamento & purificação , Vírus Auxiliares/isolamento & purificação
6.
Nature ; 617(7961): 555-563, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36996873

RESUMO

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.


Assuntos
Infecções por Adenovirus Humanos , Dependovirus , Hepatite , Criança , Humanos , Doença Aguda/epidemiologia , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/virologia , Alelos , Estudos de Casos e Controles , Linfócitos T CD4-Positivos/imunologia , Coinfecção/epidemiologia , Coinfecção/virologia , Dependovirus/isolamento & purificação , Predisposição Genética para Doença , Vírus Auxiliares/isolamento & purificação , Hepatite/epidemiologia , Hepatite/genética , Hepatite/virologia , Hepatócitos/virologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Fígado/virologia
7.
ACS Synth Biol ; 11(10): 3285-3295, 2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-36219557

RESUMO

Recombinant adeno-associated viruses (rAAV) are important gene delivery vehicles for gene therapy applications. Their production relies on plasmid transfection or virus infection of producer cells, which pose a challenge in process scale-up. Here, we describe a template for a transfection-free, helper virus-free rAAV producer cell line using a synthetic biology approach. Three modules were integrated into HEK293 cells including an rAAV genome and multiple inducible promoters controlling the expression of AAV Rep, Cap, and helper coding sequences. The synthetic cell line generated infectious rAAV vectors upon induction. Independent control over replication and packaging activities allowed for manipulation of the fraction of capsid particles containing viral genomes, affirming the feasibility of tuning gene expression profiles in a synthetic cell line for enhancing the quality of the viral vector produced. The synthetic biology approach for rAAV production presented in this study can be exploited for scalable biomanufacturing.


Assuntos
Dependovirus , Biologia Sintética , Humanos , Dependovirus/genética , Células HEK293 , Vetores Genéticos/genética , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo
8.
Int J Mol Sci ; 23(16)2022 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-36012656

RESUMO

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.


Assuntos
RNA Satélite , RNA Viral , Variação Genética , Vírus Auxiliares/genética , Nepovirus , Doenças das Plantas/genética , Plantas/genética , RNA Satélite/genética , RNA Viral/genética , Replicação Viral/genética
9.
Viruses ; 14(1)2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-35062311

RESUMO

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported "2× genome" and the "1.2× genome" infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.


Assuntos
Boidae/virologia , Células Clonais , Coinfecção/genética , Vírus Delta da Hepatite/genética , Replicação Viral , Animais , Boidae/genética , Genoma Viral , Vírus Auxiliares/genética , Hepadnaviridae/genética , Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite D/virologia , RNA Catalítico , RNA Viral/genética , Transfecção
10.
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
11.
Viruses ; 13(9)2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34578277

RESUMO

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/genética
12.
Viruses ; 13(9)2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34578372

RESUMO

Human rotaviruses (HuRVAs) are highly important causes of acute gastroenteritis in infants and young children worldwide. A lack of reliable and reproducible reverse genetics systems for HuRVAs has limited a proper understanding of HuRVA biology and also the rational design of live-attenuated vaccines. Since the development of the first reverse genetics system for RVAs (partially plasmid-based reverse genetics system) in 2006, there have been many efforts with the goal of generating infectious recombinant HuRVAs entirely from cloned cDNAs. However, the establishment of a HuRVA reverse genetics system was very challenging until 2019. This review article provides an overview of the historical background of the recent development of long-awaited HuRVA reverse genetics systems, beginning with the generation of recombinant human-simian reassortant RVAs with the aid of a helper virus in 2006 and the generation of recombinant animal (simian) RVAs in a helper virus-free manner in 2017, and culminating in the generation of recombinant HuRVAs entirely from plasmid cDNAs in 2019. Notably, the original HuRVA reverse genetics system has already been optimized to increase the efficiency of virus generation. Although the application of HuRVA reverse genetics systems has only just been initiated, these technologies will help to answer HuRVA research questions regarding viral replication and pathogenicity that could not be addressed before, and to develop next-generation vaccines and intestine-specific rotaviral vectors.


Assuntos
Genoma Viral , Plasmídeos/genética , Genética Reversa/métodos , Rotavirus/genética , Replicação Viral/genética , Vírus Auxiliares/genética , Humanos , RNA Viral/genética , Infecções por Rotavirus/virologia , Proteínas não Estruturais Virais/genética
13.
Viruses ; 13(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201626

RESUMO

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 Viral
14.
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
15.
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
16.
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
17.
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
18.
Mol Ther ; 29(5): 1808-1820, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33571680

RESUMO

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 Xenoenxerto
19.
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
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...