Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus

doi:10.3969/j.issn.1004-1524.2022.07.06

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (7): 1396-1401.DOI: 10.3969/j.issn.1004-1524.2022.07.06

• Animal Science • Previous Articles Next Articles

Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus

LIU Zhiyi(), MI Xiaoyun, HUANG Cong, LI Chuanfeng, CHEN Zongyan()

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

Received:2020-11-12 Online:2022-07-25 Published:2022-07-26
Contact: CHEN Zongyan

Abstract

Abstract:

Translocation-associated membrane protein 1 (TRAM1) is a host protein which is associated membrane protein 1, found within the endoplasmic reticulum (ER). Studies have shown that TRAM1 can relieve the pressure of endoplasmic reticulum (ER) during stress by increasing its content. Previous studies found that TRAM1 had been changed in duck reovirus infected duck embryo fibroblasts cells. In this study, the interaction between DRV non-structural protein σNS and TRAM1 had been further confirmed experimentally. σNS protein was expressed firstly. Glutathione S-transferase pull-down was utilized to confirm the interaction between TRAM1 and σNS protein extracellular while co-immunoprecipitation (Co-IP) was utilized to confirm the interaction between TRAM1 and σNS protein intracellular. In addition, knockdown of TRAM1 using siRNA and overexpression of TRAM1 gene were conducted to explore its effect on virus replication. The interaction between σNS and TRAM1 has been confirmed. The results showed that TRAM1 overexpression degraded the levels of σNS transcription while TRAM1 silencing enhanced the levels of σNS transcription. In conclusion, our studies could afford help to explore the function of σNS protein on DRV replication. It also provided data to explore the role of interactions between TRAM1 and σNS during DRV infection.

Key words: duck reovirus, σNS, virus-host interaction, TRAM1

CLC Number:

S858.32

LIU Zhiyi, MI Xiaoyun, HUANG Cong, LI Chuanfeng, CHEN Zongyan. Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1396-1401.

Figures/Tables 6

Table 1 qRT-PCR primer sequence

基因Gene	上游引物Forward primer (5'→3')	下游引物Reverse primer(5'→3')
σNS	GTTCCTTTCGACCACGTCTATC	CATGAGACGAGAGACAGCATTAG
β-actin	CCTCTATGCCAACACAGTGC	CCTGCTTGCTGATCCACATC

Fig.1 Inducible expression of GST and GST-σNS M, Protein marker; GST, GST no-load control with the final concentration of IPTG was 0.2 mmol·L-1; GST-σNS-1, GST-σNS was induced at the final IPTG concentration of 0.5 mmol·L-1; GST-σNS-2, GST-σNS was induced at the final IPTG concentration of 0.2 mmol·L-1.

Fig.2 Optimization design of inducing conditions for pGEX-4T-1-σNS expression S, Supernatant from inducible expression; I, Precipitation from inducible expression.

Fig.3 Analysis of interaction between σNS protein and TRAM1 protein by Co-IP

Fig.4 Analysis of the interaction between σNS protein and TRAM1 protein by GST pull-down

Fig.5 Effects of TRAM1 protein on DRV-TH11 replication A, Western blot was used to detect the effect of siRNA knockdown on TRAM1; B, The mRNA level of DRV-TH11 σNS gene was detected by using qRT-PCR after knocking down TRAM1; C, The mRNA level of DRV-TH11 σNS gene was detected by using qRT-PCR after upregulating TRAM1. ****, P<0.001; ####, P<0.000 1.

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Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus

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