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1.
J Virol ; 96(15): e0198021, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35852352

RESUMO

Atypical porcine pestiviruses (APPV; Pestivirus K) are a recently discovered, very divergent species of the genus Pestivirus within the family Flaviviridae. The presence of APPV in piglet-producing farms is associated with the occurrence of so-called "shaking piglets," suffering from mild to severe congenital tremor type A-II. Previous studies showed that the cellular protein DNAJC14 is an essential cofactor of the NS2 autoprotease of all classical pestiviruses. Consequently, genetically engineered DNAJC14 knockout cell lines were resistant to all tested noncytopathogenic (non-cp) pestiviruses. Surprisingly, we found that the non-cp APPV can replicate in these cells in the absence of DNAJC14, suggesting a divergent mechanism of polyprotein processing. A complete laboratory system for the study of APPV was established to learn more about the replication of this unusual virus. The inactivation of the APPV NS2 autoprotease using reverse genetics resulted in nonreplicative genomes. To further investigate whether a regulation of the NS2-3 cleavage is also existing in APPV, we constructed synthetic viral genomes with deletions and duplications leading to the NS2 independent release of mature NS3. As observed with other pestiviruses, the increase of mature NS3 resulted in elevated viral RNA replication levels and increased protein expression. Our data suggest that APPV exhibit a divergent mechanism for the regulation of the NS2 autoprotease activity most likely utilizing a different cellular protein for the adjustment of replication levels. IMPORTANCE DNAJC14 is an essential cofactor of the pestiviral NS2 autoprotease, limiting replication to tolerable levels as a prerequisite for the noncytopathogenic biotype of pestiviruses. Surprisingly, we found that the atypical porcine pestivirus (APPV) is able to replicate in the absence of DNAJC14. We further investigated the NS2-3 processing of APPV using a molecular clone, monoclonal antibodies, and DNAJC14 knockout cells. We identified two potential active site residues of the NS2 autoprotease and could demonstrate that the release of NS3 by the NS2 autoprotease is essential for APPV replication. Defective interfering genomes and viral genomes with duplicated NS3 sequences that produce mature NS3 independent of the NS2 autoprotease activity showed increased replication and antigen expression. It seems likely that an alternative cellular cofactor controls NS2-3 cleavage and thus replication of APPV. The replication-optimized synthetic APPV genomes might be suitable live vaccine candidates, whose establishment and testing warrant further research.


Assuntos
Chaperonas Moleculares , Infecções por Pestivirus , Pestivirus , Suínos , Replicação Viral , Animais , Linhagem Celular , Coenzimas , Genoma Viral/genética , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/genética , Pestivirus/classificação , Pestivirus/enzimologia , Pestivirus/crescimento & desenvolvimento , Infecções por Pestivirus/veterinária , RNA Viral/genética , Suínos/virologia , Doenças dos Suínos/virologia , Proteases Virais/metabolismo , Replicação Viral/genética
2.
Viruses ; 13(5)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922699

RESUMO

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.


Assuntos
Drosophila/virologia , Espécies Introduzidas , Vírus de RNA/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Vírus de RNA/ultraestrutura , RNA Viral , Análise de Sequência de RNA , Carga Viral , Proteínas Estruturais Virais/química
3.
RNA Biol ; 18(10): 1445-1457, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33258405

RESUMO

Anoxygenic photosynthesis is an important pathway for Rhodobacter sphaeroides to produce ATP under oxygen-limiting conditions. The expression of its photosynthesis genes is tightly regulated at transcriptional and post-transcriptional levels in response to light and oxygen signals, to avoid photooxidative stress by the simultaneous presence of pigments, light and oxygen. The puf operon encodes pigment-binding proteins of the light-harvesting complex I (genes pufB and pufA), of the reaction centre (genes pufL and pufM), a scaffold protein (gene pufX) and includes the gene for sRNA PcrX. Segmental differences in the stability of the pufBALMX-pcrX mRNA contribute to the stoichiometry of LHI to RC complexes. With asPcrL we identified the third sRNA and the first antisense RNA that is involved in balancing photosynthesis gene expression in R. sphaeroides. asPcrL influences the stability of the pufBALMX-pcrX mRNA but not of the pufBA mRNA and consequently the stoichiometry of photosynthetic complexes. By base pairing to the pufL region asPcrL promotes RNase III-dependent degradation of the pufBALMX-prcX mRNA. Since asPcrL is activated by the same protein regulators as the puf operon including PcrX it is part of an incoherent feed-forward loop that fine-tunes photosynthesis gene expression.[Figure: see text].


Assuntos
Genes Bacterianos , Complexo de Proteínas do Centro de Reação Fotossintética/genética , RNA Antissenso/genética , Rhodobacter sphaeroides/fisiologia , Ribonuclease III/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Complexos de Proteínas Captadores de Luz/genética , Óperon , Fotossíntese , RNA Bacteriano/genética , Rhodobacter sphaeroides/genética
4.
Life Sci Alliance ; 1(4): e201800080, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30456366

RESUMO

Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5' RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.

5.
Mol Microbiol ; 110(3): 325-334, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29995316

RESUMO

Facultative phototrophic bacteria like Rhodobacter sphaeroides can produce ATP by anoxygenic photosynthesis, which is of advantage under conditions with limiting oxygen. However, the simultaneous presence of pigments, light and oxygen leads to the generation of harmful singlet oxygen. In order to avoid this stress situation, the formation of photosynthetic complexes is tightly regulated by light and oxygen signals. In a complex regulatory network several regulatory proteins and the small non-coding RNA PcrZ contribute to the balanced expression of photosynthesis genes. With PcrX this study identifies a second sRNA that is part of this network. The puf operon encodes pigment binding proteins of the light-harvesting I complex (PufBA) and of the reaction center (PufLM), a protein regulating porphyrin flux (PufQ), and a scaffolding protein (PufX). The PcrX sRNA is derived from the 3' UTR of the puf operon mRNA by RNase E-mediated cleavage. It targets the pufX mRNA segment, reduces the half-life of the pufBALMX mRNA and as a consequence affects the level of photosynthetic complexes. By its action PcrX counteracts the increased expression of photosynthesis genes that is mediated by protein regulators and is thus involved in balancing the formation of photosynthetic complexes in response to external stimuli.


Assuntos
Regiões 3' não Traduzidas , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Complexos de Proteínas Captadores de Luz/biossíntese , Fotossíntese , Pequeno RNA não Traduzido/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Complexos de Proteínas Captadores de Luz/genética , Óperon , Pequeno RNA não Traduzido/genética , Rhodobacter sphaeroides/genética
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