Yuan Shishan, Murtaugh Michael P, Schumann Faith A, Mickelson Dan, Faaberg Kay S
Department of Veterinary PathoBiology, University of Minnesota, 205 Veterinary Science Building, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA.
Virus Res. 2004 Sep 15;105(1):75-87. doi: 10.1016/j.virusres.2004.04.015.
In this study, porcine reproductive and respiratory syndrome virus (PRRSV) heteroclite (uncommon forms) RNAs were characterized. Nucleotide sequencing of 11 additional defective RNA species verified that heteroclites are formed between the 5' and 3' termini of PRRSV at short stretches of identity, with variability seen between the junction sites utilized. Northern blot and RT-PCR analyses indicated that heteroclite RNA species were likely to be packaged into purified virions. To study whether heteroclite RNAs and viral genomic RNAs could be packaged into the same virions, PRRSV strain VR-2332 was purified by sucrose density gradient centrifugation. RT-PCR amplification of the viral RNAs isolated from three distinct gradient bands, using genomic- and heteroclite-specific primer pairs, demonstrated that heteroclite RNAs could not be readily dissociated from genomic RNA. Partial segregation of full-length and larger heteroclite genomes to the upper two gradient bands was seen, but smaller species could be found in all three fractions. These results strongly suggest that heteroclite RNAs retain the PRRSV RNA packaging signal. In vitro transcription and translation of one heteroclite cDNA clone verified that the RNA could express a predicted 32.6 kDa protein, indicating that these RNA species have the potential to produce abnormal proteins in infected cells.
在本研究中，对猪繁殖与呼吸综合征病毒（PRRSV）异形（罕见形式）RNA进行了表征。对另外11种缺陷RNA种类的核苷酸测序证实，异形RNA是在PRRSV的5'和3'末端之间短片段的相同序列处形成的，不同的连接位点存在变异性。Northern印迹和RT-PCR分析表明，异形RNA种类可能被包装到纯化的病毒粒子中。为了研究异形RNA和病毒基因组RNA是否能被包装到同一病毒粒子中，通过蔗糖密度梯度离心法纯化了PRRSV毒株VR-2332。使用基因组特异性和异形特异性引物对，对从三个不同梯度条带中分离的病毒RNA进行RT-PCR扩增，结果表明异形RNA不易与基因组RNA分离。全长和较大的异形基因组部分分离到上部两个梯度条带中，但在所有三个组分中都能发现较小的种类。这些结果强烈表明异形RNA保留了PRRSV RNA包装信号。对一个异形cDNA克隆进行体外转录和翻译，证实该RNA能表达预测的32.6 kDa蛋白，表明这些RNA种类有可能在感染细胞中产生异常蛋白。
