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1.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32759316

RESUMO

An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low-replicating attenuated vaccine candidates or low-cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.


Assuntos
Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vírus Reordenados/genética , Genética Reversa/métodos , Rotavirus/genética , Proteínas Virais/genética , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Animais , Chlorocebus aethiops , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Camundongos , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Capuzes de RNA , Vírus Reordenados/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Rotavirus/imunologia , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transfecção , Células Vero , Proteínas Virais/imunologia , Replicação Viral
2.
Nucleic Acids Res ; 47(5): 2681-2698, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30726994

RESUMO

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.


Assuntos
Núcleo Celular/genética , Citoplasma/genética , RNA Polimerases Dirigidas por DNA/genética , Transcrição Gênica , Animais , Células CHO , Cricetulus , Citoplasma/química , RNA Polimerases Dirigidas por DNA/química , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Humanos , Poli A/genética , Poliadenilação/genética
3.
J Virol ; 91(11)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28298603

RESUMO

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid µ1 protein to µ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation.IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.


Assuntos
Vírus da Febre Suína Africana/enzimologia , Nucleotidiltransferases/metabolismo , Orthoreovirus de Mamíferos/genética , Orthoreovirus de Mamíferos/fisiologia , RNA Viral/metabolismo , Vírion/fisiologia , Montagem de Vírus , Vírus da Febre Suína Africana/genética , Linhagem Celular , Proteínas Recombinantes de Fusão/metabolismo , Genética Reversa , Vírion/genética , Replicação Viral
4.
Sci Rep ; 14(1): 5156, 2024 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-38431749

RESUMO

We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3'UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5-3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.


Assuntos
RNA Polimerases Dirigidas por DNA , Poliadenilação , Animais , Humanos , Camundongos , RNA Polimerases Dirigidas por DNA/genética , RNA Mensageiro/metabolismo , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Poli A/genética , Poli A/metabolismo
5.
Genet Med ; 7(2): 83-96, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15714075

RESUMO

It is regularly thought that human complex disorder susceptibility genes show differences in gene expression between normal and pathologic tissues. Thus, differences of transcript amounts could be indicative of complex disorder susceptibility loci and, therefore, be used for the discovery or the validation of human susceptibility genes to complex disorders/traits. Whether human complex disorder susceptibility genes effectively display differences in transcript amounts was tested by meta-analysis of the published literature comparing transcript amounts of well-validated human susceptibility genes to complex traits/disorders. A total of 94 gene-disease associations, which were studied in at least three independent studies and showed strong evidence of positive association, were analyzed. For 23 out of these 94 well-validated gene-disease associations, 120 gene expression studies comparing normal and pathologic human tissues were found. For 60 out of these 120 gene expression studies, the difference of level expression between normal and pathologic human tissues was statistically significant. This result was highly significant, as only 6 significant results were expected randomly under the null hypothesis (P < 10(-112)). A large excess of replication studies were also found, which were in agreement with the original report (P = 6 x 10(-4)). However, the overall level of expression change between normal and pathologic human tissues was relatively moderate, because only 36 (60%) and 19 (31.6%) out of the 62 statistically significant gene expression studies reached 2- or 3-fold changes in expression level, respectively. The present meta-analysis confirms statistical differences of expression levels between normal and pathologic human tissues for human susceptibility genes to complex traits/disorders. However, the levels of differences in transcript amounts appear to be relatively weak. These findings rationalize the use of gene expression for the discovery/validation of human susceptibility genes, but the weak differences of expression typically found should be taken into account for the design of such studies.


Assuntos
Regulação da Expressão Gênica , Predisposição Genética para Doença , Doenças Genéticas Inatas , Humanos , Locos de Características Quantitativas , RNA/biossíntese
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