干扰和稳定表达GP5对猪繁殖与呼吸综合征病毒感染早期复制的影响

doi:10.11843/j.issn.0366-6964.2018.06.015

Abstract

Abstract:

To investigate the effect of GP5, the most important structural protein encoded by porcine reproductive and respiratory syndrome virus (PRRSV) ORF5, on virus replication and it's mechanism, a recombinant plasmid (named as pPB-GP5) was constructed using PiggyBac Transposon System Vectors and transfected into Marc-145 cells. Marc-145 cell line stably expressing GP5 was obtained by puromycin resistance screening and three times sub-cloning. GP5 was confirmed stable expression in Marc-145 cells by RT-PCR, Western blot and IFA detection and the cells was named as Marc-145-GP5. On the basis that GP5 expression does not affect cell proliferation, the effect of GP5 stable expression on virus replication was investigated by N gene copies, N protein expression, and viral titers in supernatant after PRRSV SD16 infecting Marc-145-GP5 cells. IFN-α, IFN-β, and IFN-γ protein levels were analyzed using monkey IFN ELISA kit. Then three pairs specific small interfering RNA (siRNA), siRNA GP5-471, siRNA GP5-404 and siRNA GP5-525, targeting ORF5 and one pair negative control siRNA, scrambled siRNA, were designed and synthesized. siRNAs were transfected into Marc-145 cells and then the cells were infected with 0.1 multiplicity of infection (MOI) PRRSV SD16 strain at 6 h or 12 h after transfection. The cells were collected at 24 h after infection and total RNA or protein were extracted. GP5 mRNA level was detected using real time PCR (qPCR) and N protein level was analyzed using Western blot, respectively. RT-PCR, Western bolt and IFA detection results showed that GP5 was stably expressed in Marc-145 cells and did not affect cell proliferation. But GP5 expression in Marc-145 cells promoted the replication of PRRSV in early infection time through down-regulating of IFN production. Transfection with specific siRNAs targeting ORF5 inhibited PRRSV replication and siRNA GP5-471, at a concentration of 100 nmol·L^-1, showed the most effective inhibition and had highly significant difference compared to negative control siRNA treated group. These findings suggest that GP5 plays a regulatory role in PRRSV replication.

Key words: porcine reproductive and respiratory syndrome virus, GP5, small interfering RNA (siRNA), stable expression, interferon

CLC Number:

S852.659.6

SONG Lin-lin, JIA Cun-yu, HAN Xi-meng, ZHOU En-min, MU Yang. The Effect of Interfering and Stable Expression of GP5 on the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Early Infection[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1231-1240.

References

[1] RAHE M C,MURTAUGH M P.Mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus[J].Viruses,2017,9(6):148.
[2] GUO Z H,CHEN X X,LI R,et al.The prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in China:a molecular epidemiological perspective[J].Virol J,2018,15(1):2.
[3] LUNNEY J K,FANG Y,LADINIG A,et al.Porcine reproductive and respiratory syndrome virus (PRRSV):pathogenesis and interaction with the immune system[J].Annu Rev Anim Biosci,2016,4:129-154.
[4] HAN J,ZHOU L,GE X N,et al.Pathogenesis and control of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J].Vet Microbiol,2017,209:30-47.
[5] DU T F,NAN Y C,XIAO S Q,et al.Antiviral strategies against PRRSV infection[J].Trends Microbiol,2017,25(12):968-979.
[6] NAN Y C,WU C Y,GU G Q,et al.Improved vaccine against PRRSV:current progress and future perspective[J].Front Microbiol,2017,8:1635.
[7] DE LOS SANTOS T,WU Q H,DE AVILA BOTTON S,et al.Short hairpin RNA targeted to the highly conserved 2B nonstructural protein coding region inhibits replication of multiple serotypes of foot-and-mouth disease virus[J].Virology,2005,335(2):222-231.
[8] DURYMANOVA ONO E A,IAMAMOTO K,CASTILHO J G,et al.In vitro and in vivo inhibition of rabies virus replication by RNA interference[J].Braz J Microbiol,2013,44(3):879-882.
[9] LI L,LI Q Y,BAO Y H,et al.RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs[J].J Biotechnol,2014,171:17-24.
[10] CHEN Y H,WANG Y,ZENG K,et al.Knockdown expression of IL-10Rα gene inhibits PRRSV replication and elevates immune responses in PBMCs of Tibetan pig in vitro[J].Vet Res Commun,2018,42(1):11-18,doi:10.1007/s11259-017-9703-z.
[11] CHUNG N P,MATTHEWS K,KIM H J,et al.Stable 293 T and CHO cell lines expressing cleaved,stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies[J].Retrovirology,2014,11:33.
[12] 王晓红,宋林林,李亮亮,等.GP5^Δ84-119稳定表达对猪繁殖与呼吸综合征病毒复制的影响[J].畜牧兽医学报,2016,47(2):315-324.
WANG X H,SONG L L,LI L L,et al.The effect of GP5^Δ84-119 stable expression on the replication of porcine reproductive and respiratory syndrome virus[J].Acta Veterinaria et Zootechnica Sinica,2016,47(2):315-324.(in Chinese)
[13] MU Y,LI L L,ZHANG B B,et al.Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest,but does not induce cellular apoptosis in Marc-145 cells[J].Virology,2015,484:136-145.
[14] HONG H X,ZHANG Y M,XU H,et al.Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase[J].Mol Cells,2007,24(3):358-363.
[15] FENG L L,ZHANG X Y,XIA X L,et al.Generation and characterization of a porcine endometrial endothelial cell line susceptible to porcine reproductive and respiratory syndrome virus[J].Virus Res,2013,171(1):209-215.
[16] 穆杨,李亮亮,刘园园,等.PRRSV感染对猪肺泡巨噬细胞Bax和Bcl-2 mRNA表达的影响[J].中国兽医学报,2013,33(9):1313-1318,1351.
MU Y,LI L L,LIU Y Y,et al.Bax and Bcl-2 gene expression in porcine alveolar macrophages infected by porcine reproductive and respiratory syndrome virus[J].Chinese Journal of Veterinary Science,2013,33(9):1313-1318,1351.(in Chinese)
[17] GAGNON C A,LACHAPELLE G,LANGELIER Y,et al.Adenoviral-expressed GP₅ of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions[J].Arch Virol,2003,148(5):951-972.
[18] WANG F X,WEN Y J,YANG B C,et al.Role of non-structural protein 2 in the regulation of the replication of the porcine reproductive and respiratory syndrome virus in MARC-145 cells:effect of gene silencing and over expression[J].Vet Microbiol,2012,161(1-2):58-65.
[19] GAO J M,JI P C,ZHANG M D,et al.GP5 expression in Marc-145 cells inhibits porcine reproductive and respiratory syndrome virus infection by inducing beta interferon activity[J].Vet Microbiol,2014,174(3-4):409-418.
[20] SUR J H,DOSTER A R,OSORIO F A.Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus[J].Vet Pathol,1998,35(6):506-514.
[21] SIRINARUMITR T,ZHANG Y,KLUGE J P,et al.A pneumo-virulent United States isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo[J].J Gen Virol,1998,79(Pt 12):2989-2995.
[22] FERNÁNDEZ A,SUÁREZ P,CASTRO J M,et al.Characterization of regions in the GP₅ protein of porcine reproductive and respiratory syndrome virus required to induce apoptotic cell death[J].Virus Res,2002,83(1-2):103-118.
[23] LEE C,ROGAN D,ERICKSON L,et al.Characterization of the porcine reproductive and respiratory syndrome virus glycoprotein 5(GP5) in stably expressing cells[J].Virus Res,2004,104(1):33-38.
[24] WANG X P,WEI R F,LI Q Y,et al.PK-15 cells transfected with porcine CD163 by PiggyBac transposon system are susceptible to porcine reproductive and respiratory syndrome virus[J].J Virol Methods,2013,193(2):383-390.
[25] YIN J,BI X B.Generation and characterization of virus-free reprogrammed melanoma cells by the piggyBac transposon[J].J Cancer Res Clin Oncol,2013,139(9):1591-1599.
[26] LI R B,ZHUANG Y,HAN M,et al.piggyBac as a high-capacity transgenesis and gene-therapy vector in human cells and mice[J].Dis Model Mech,2013,6(3):828-833.
[27] YUAN S Z,ZHANG N,XU L,et al.Induction of apoptosis by the nonstructural protein 4 and 10 of porcine reproductive and respiratory syndrome virus[J].PLoS One,2016,11(6):e0156518.
[28] BROCKMEIER S L,LOVING C L,EBERLE K C,et al.Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine[J].Vet Microbiol, 2017, 212:48-51.
[29] ZHAO M, WAN B, LI H, et al. Porcine 2', 5'-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro[J]. Microb Pathog, 2017, 111:14-21.

The Effect of Interfering and Stable Expression of GP5 on the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Early Infection

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