PMID- 29969475
OWN - NLM
STAT- MEDLINE
DCOM- 20190107
LR  - 20190107
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 13
IP  - 7
DP  - 2018
TI  - Utilizing host endogenous microRNAs to negatively regulate the replication of 
      porcine reproductive and respiratory syndrome virus in MARC-145 cells.
PG  - e0200029
LID - 10.1371/journal.pone.0200029 [doi]
LID - e0200029
AB  - 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.
FAU - Li, Liwei
AU  - Li L
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
FAU - Gao, Fei
AU  - Gao F
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
AD  - Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal 
      Infectious Disease and Zoonose, Yangzhou University, Yangzhou, PR China.
FAU - Zheng, Hao
AU  - Zheng H
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
AD  - Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal 
      Infectious Disease and Zoonose, Yangzhou University, Yangzhou, PR China.
FAU - Jiang, Yifeng
AU  - Jiang Y
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
AD  - Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal 
      Infectious Disease and Zoonose, Yangzhou University, Yangzhou, PR China.
FAU - Tong, Wu
AU  - Tong W
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
AD  - Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal 
      Infectious Disease and Zoonose, Yangzhou University, Yangzhou, PR China.
FAU - Zhou, Yanjun
AU  - Zhou Y
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
AD  - Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal 
      Infectious Disease and Zoonose, Yangzhou University, Yangzhou, PR China.
FAU - Tong, Guangzhi
AU  - Tong G
AUID- ORCID: 0000-0001-7048-0837
AD  - Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 
      Shanghai, PR China.
AD  - Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal 
      Infectious Disease and Zoonose, Yangzhou University, Yangzhou, PR China.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20180703
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (MicroRNAs)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Host-Pathogen Interactions/*genetics
MH  - MicroRNAs/*genetics
MH  - Mutation
MH  - Porcine respiratory and reproductive syndrome virus/*physiology
MH  - Transcription, Genetic
MH  - Virus Replication/*genetics
PMC - PMC6029797
COIS- The authors have declared that no competing interests exist.
EDAT- 2018/07/04 06:00
MHDA- 2019/01/08 06:00
PMCR- 2018/07/03
CRDT- 2018/07/04 06:00
PHST- 2018/02/11 00:00 [received]
PHST- 2018/06/17 00:00 [accepted]
PHST- 2018/07/04 06:00 [entrez]
PHST- 2018/07/04 06:00 [pubmed]
PHST- 2019/01/08 06:00 [medline]
PHST- 2018/07/03 00:00 [pmc-release]
AID - PONE-D-18-04611 [pii]
AID - 10.1371/journal.pone.0200029 [doi]
PST - epublish
SO  - PLoS One. 2018 Jul 3;13(7):e0200029. doi: 10.1371/journal.pone.0200029. 
      eCollection 2018.