Rab32对禽偏肺病毒C型复制的影响

doi:10.11843/j.issn.0366-6964.2024.09.028

Abstract

Abstract:

To investigate the effect of Rab32 on the replication of avian metapneumovirus type C (aMPV/C), confocal images were obtained and co-localization coefficients were analyzed by Image J software after infected with A549 cells by aMPV/C. Furthermore, the plasmids of pCMV-Flag-Rab32 or pCMV-Flag as well as siRab32 or siNC RNA sequences were transfected into A549 cells before aMPV/C infection, respectively. The transcription level of aMPV/C N gene, the expression level of aMPV/C N and Rab32 proteins, and the viral titers in collected samples were detected by quantitative real-time PCR (qPCR), Western blot, and the half tissue culture infective dose (TCID₅₀) method, respectively. Then, the plasmids of pCMV-mcherry-Rab32 and GFP-tagged aMPV/C proteins were co-transfected into A549 cells before conducting confocal imaging, respectively. Subsequently, the plasmids of pCMV-Flag-Rab32 and GFP-tagged aMPV/C proteins were co-transfected into HEK293T cells before collecting cell samples at 36 h post-transfection and conducting the co-immunoprecipitation assay. The results showed that obvious co-localized fluorescent signals of Rab32 and aMPV/C were observed in the cytoplasm. The co-localization coefficients of Rab32 and N proteins were significantly higher than that of mock-infected group. The transcription level of the N gene, the expression level of Rab32 and N proteins, and the viral titers were significantly increased (P<0.01) after overexpression of Rab32, respectively. On the contrary, the transcription level of the N gene, the expression level of Rab32 and N proteins, and the viral titers were significantly decreased (P<0.01) after knockdown expression of Rab32, respectively. Obvious co-localized fluorescent signals of Rab32 and aMPV/C proteins (N, F, M, G, SH, P, M2-1, M2-2, L1, L2, and L4) were observed in the cytoplasm, while scarcely co-localized signals of Rab32 and L3 protein were found. Specific bands of N and M2-2 proteins were found in immunoprecipitation samples while no bands of other GFP-tagged aMPV/C proteins were found. These results indicating that Rab32 interacts with aMPV/C N and M2-2 proteins. In summary, Rab32 might affect aMPV/C replication by interacting with multiple viral proteins (N and M2-2). The relevant results can provide the basis for elucidating the mechanism of Rab32 regulating aMPV/C replication via protein interaction.

Key words: Rab32, avian metapneumovirus type c, replication, expression level, interaction

CLC Number:

S852.659.5

Xufei FENG, Yuxiang QI, Hanzhe YU, Zhengzhou ZHANG, Rujia WANG, Chuang MENG, Kui DONG. Effect of Rab32 on the Replication of Avian Metapneumovirus Type C[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4041-4050.

Figures/Tables 6

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References 24

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引物名称 Primer name	序列(5′→3′) Sequences
aMPV/C-N F	GAGGTTCCCGAGGATTGACA
aMPV/C-N R	GATCACTCCCCACCTTAGCA
GAPDH F	GACAGTCAGCCGCATCTTCT
GAPDH R	GCGCCCAATACGACCAAATC

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Effect of Rab32 on the Replication of Avian Metapneumovirus Type C

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