PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein is the most abundant structural protein of the virus. During infection, the N protein is specifically localized to the nucleus and nucleolus in addition to its normal cytoplasmic distribution. Previously, a nuclear localization signal (NLS, 41-PGKK(N/S)KKKN)-null mutant virus (41-PGGGNKKKN) showed reduced viremia and increased production of neutralizing antibodies in infected pigs. However, the mutagenized NLS underwent strong selection pressure in the pig that resulted in partial or complete reversion and reacquisition of NLS function, and thus the biological effect of the NLS-null mutation needed further investigation. In the present study, a total of 9 "reversion resistant" mutants were generated by amino acid deletions and substitutions using an infectious cDNA clone. Two mutant clones (PG--SKKKS and PG--S-KKS) that produced progeny viruses were genetically stable for at least 20 passages in cell culture. Infection of pigs with those mutants induced neutralizing antibodies to higher titers than with wild-type virus. Both mutant viruses induced viremia of lower titer and of shorter duration than wild-type virus. RT-PCR from tonsils showed that both mutants persisted at a reduced level. Virus transmission to contact pigs was also lower in the mutant virus infected groups. No reversion to functional NLS was detected in either mutant from any pig. These data demonstrate that N protein nuclear localization is indeed associated with viral pathogenesis and host response to PRRS.

译文

:PRRSV(猪生殖和呼吸综合征病毒)核衣壳(N)蛋白是该病毒中最丰富的结构蛋白。在感染过程中,N蛋白除了其正常的细胞质分布外,还特异地位于细胞核和核仁中。以前,核定位信号(NLS,41-PGKK(N / S)KKKN)-无效突变病毒(41-PGGGNKKKN)显示出降低的病毒血症,并增加了感染猪中和抗体的产生。但是,诱变的NLS在猪中受到强大的选择压力,导致NLS功能部分或完全回复和重新获得,因此NLS-null突变的生物学效应需要进一步研究。在本研究中,使用感染性cDNA克隆通过氨基酸缺失和置换产生了总共9个“抗逆转”突变体。产生后代病毒的两个突变体克隆(PG--SKKKS和PG--S-KKS)在细胞培养中至少20代具有遗传稳定性。用那些突变体感染猪所诱导的中和抗体的滴度要高于野生型病毒。与野生型病毒相比,两种突变病毒均能引起较低的滴度和持续时间较短的病毒血症。扁桃体的RT-PCR显示两个突变体均以降低的水平持续存在。在突变病毒感染组中,接触猪的病毒传播率也较低。在任何猪的任一突变体中均未检测到功能性NLS的逆转。这些数据表明,N蛋白核定位确实与病毒发病机制和宿主对PRRS的反应有关。

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