Utilizing host endogenous microRNAs to negatively regulate the replication of porcine reproductive and respiratory syndrome virus in MARC-145 cells

Autor: Liwei Li, Wu Tong, Fei Gao, Yan-Jun Zhou, Hao Zheng, Guangzhi Tong, Yi-Feng Jiang
Jazyk: angličtina
Rok vydání: 2018
Předmět:
0301 basic medicine
Untranslated region
Transcription
Genetic
lcsh:Medicine
Virus Replication
Biochemistry
Transcription (biology)
lcsh:Science
Regulation of gene expression
Viral Genomics
Insertion Mutation
Multidisciplinary
biology
Microbial Mutation
Genomics
Cell biology
Nucleic acids
Viral evolution
Host-Pathogen Interactions
Viral Genome
Research Article
Nucleotide Sequencing
Microbial Genomics
Transfection
Research and Analysis Methods
Microbiology
Deep sequencing
Cell Line
03 medical and health sciences
Virology
Genetics
Animals
Point Mutation
Gene silencing
Porcine respiratory and reproductive syndrome virus
Non-coding RNA
Molecular Biology Techniques
Sequencing Techniques
Molecular Biology
Biology and life sciences
lcsh:R
Porcine reproductive and respiratory syndrome virus
biology.organism_classification
Viral Replication
Gene regulation
MicroRNAs
030104 developmental biology
Viral replication
Mutation
RNA
lcsh:Q
Gene expression
Zdroj: PLoS ONE, Vol 13, Iss 7, p e0200029 (2018)
PLOS ONE
PLoS ONE
ISSN: 1932-6203
Popis: MicroRNAs (miRNAs) contribute to gene regulation at the post-transcriptional level and are capable of mRNA silencing by binding to target sites exhibiting high degrees of complementarity. Therefore, cloning host miRNA-recognition sequences into the genome of RNA viruses represents a rational strategy for manipulating viral replication. Here, we performed deep sequencing to obtain small-RNA (sRNA)-expression profiles from in vitro-cultured MARC-145 cells post infection with porcine reproductive and respiratory syndrome virus (PRRSV) and chose six candidate miRNAs of different abundance (miR-21, miR-140-3p, miR-185, miR-26a, miR-505, and miR-199a) for further study. Based on the full-length cDNA clone p7USC, we constructed a number of PRRSV mutants that provided complementary base-pairing target sites for the miRNAs in 3′ untranslated regions. Our results showed that all low- and moderate- abundant miRNA-target mutants showed similar growth properties, whereas the highest-abundant miRNA-target mutant blocked both viral transcription and replication. Discontinuous mutations in high-abundant miRNA-target sites subsequently recovered viral viability and propagation. These results demonstrated the copy number of endogenous miRNAs and the extent of sRNA complementarity were key factors to silence potential mRNA expression/translation, thereby determining PRRSV viability. Interestingly, the mutant containing miR-140-target sites (v140-t) showed strong suppression of viral replication from P1 to P3 in vitro, as shown by virus titer, plaque morphology, and qRT-PCR assays. To assess genetic stability, sequencing of v140-t (P1, P3, P5 and P10) revealed spontaneous mutations preferentially located among several nucleotides near the 3′ end of the insertion region and corresponding to the “seed region” of miR-140-3p, explaining the induced viral repression and the direction of virus evolution. This approach provided a general silencing strategy for limiting PRRSV replication by endogenous miRNAs in MARC-145 cells.
Databáze: OpenAIRE
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