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.
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.
复制标题
DOI：
10.1007/s00705-002-0943-y
复制
发表时间：
2003-05
影响因子：
2.7
通讯作者：
Dea S
中科院分区：
文献类型：
--
作者：
Gagnon CA;Lachapelle G;Langelier Y;Massie B;Dea S
利用复制缺陷型人5型腺病毒载体（hAdVs）克隆并表达了PRRSV IAF-Klop株3个主要结构蛋白GP5、M和N的orf5、orf6和orf7。虽然M蛋白基因可以克隆到hAdVs中，并在hCMV即时早期启动子/增强子的控制下在293细胞中组成表达，但表达N和GP5蛋白的hAdVs似乎是有毒的或干扰腺病毒复制，只能通过在转移载体pAdTR5中包含四环素可调节的启动子来产生。重组蛋白（rec）在分子量和抗原性方面与原病毒蛋白相似。然而，recGP5显然具有不同的n链寡糖残基。它对内切β-半乳糖苷酶的敏感性表明，聚n -乙酰乳胺存在于个体表达的蛋白上，而不存在于锚定在病毒粒子包膜上的真实GP5上。显然，recp5作为一种糖蛋白在内质网室中积累，具有高甘露糖n -连接的低聚糖侧链，对内do-β- n -乙酰氨基葡萄糖酶H处理敏感，相比之下，其病毒对应物的n -连接的低聚糖侧链同时具有高甘露糖和复杂类型。与prrsv感染的细胞一样，表达M和GP5的hAdVs共同感染293细胞不会导致M-GP5异源二聚体的形成。此外，用可诱导的hav /ORF5感染的细胞显示，北美菌株的GP5是促凋亡的。事实上，当打开表达盒时，hav /ORF5感染细胞中的caspase 3活性增强，并且可以通过TUNEL检测DNA片段。Western blot和病毒中和试验显示，经两次皮内注射hav /ORF5的猪，在用同源强毒PRRSV毒株攻击后10天内，就产生了针对真病毒GP5的抗体滴度，表明建立了特异性免疫记忆。
The ORFs 5, 6 and 7, encoding for the three major structural proteins, GP5, M and N, of the IAF-Klop strain of PRRSV were cloned and expressed in 293 cells using replication-defective human type 5 adenoviral vectors (hAdVs). Although the M protein gene could be cloned into hAdVs and expressed constituvely in 293 cells under the control of the hCMV immediate early promotor/enhancer, hAdVs expressing N and GP5 proteins, which appeared to be toxic or interfered with adenovirus replication, could only be generated by inclusion of a tetracycline-regulatable promotor in the transfer vector pAdTR5. The recombinant (rec) proteins appeared similar to the authentic viral proteins in regards to their M rs and antigenicities. However, the recGP5 apparently possesses different N-linked oligosaccharides residues. Its sensitivity to endo-β-galactosidase digestion indicates that poly-N-acetyllactosamine is present on the individually-expressed protein, but not on the authentic GP5 anchored into the virion envelope. The recGP5 apparently accumulates within the ER compartment as a glycoprotein that possesses high-mannose N-linked oligosaccharide side chains sensitive to endo-β-N-acetylglucosaminidase H treatment, by contrast to its viral counterpart for which N-linked oligosaccharide side chains are of both high-mannose and complex types. Coinfection of 293 cells with hAdVs expressing the M and GP5 did not lead to M-GP5 heterodimer formation, as demonstrated in PRRSV-infected cells. Moreover, cells infected with inducible hAdV/ORF5 showed that GP5 of the North American strain is proapoptotic. Indeed, when the expression cassette was turned-on, caspase 3 activity in hAdV/ORF5 infected cells was enhanced and DNA fragmentation could be detected by TUNEL assays. Pigs intradermally injected twice with hAdV/ORF5 developed antibody titers to the authentic viral GP5 as soon as 10 days following challenge with the homologous virulent PRRSV strain, as revealed by Western blot and virus neutralization tests, suggesting the establishment of a specific immune memory.
登录
查看更多内容
影响因子：
3.7
作者：
Mardassi, H;Massie, B;Dea, S
通讯作者：
Dea, S
影响因子：
5.5
作者：
Gonin, P;Oualikene, W;Eloit, M
通讯作者：
Eloit, M
影响因子：
2.2
作者：
Massie, B;Mosser, DD;Langelier, Y
通讯作者：
Langelier, Y
影响因子：
2.7
作者：
Dea S;Gagnon CA;Mardassi H;Pirzadeh B;Rogan D
通讯作者：
Rogan D
影响因子：
3.5
作者：
ACSADI, G;JANI, A;KARPATI, G
通讯作者：
KARPATI, G