Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
PLoS One. 2013 Sep 3;8(9):e74000. doi: 10.1371/journal.pone.0074000. eCollection 2013.
The p23 is a unique protein in the Hibiscus chlorotic ringspot virus which belongs to Family Tombusviridae Genus Carmovirus. Our previous results showed that the p23 is indispensable for host-specific replication and is localized in the nucleus with a novel nuclear localization signal. To investigate additional function(s) of p23, mutations of basic amino acids lysine (K), arginine (R) and histidine (H) that abolish its nuclear localization, were introduced into a biologically active full-length cDNA clone p223 of HCRSV for testing its effects on virus replication and virus movement in vivo. Primer-specific reverse transcription-PCR was conducted to detect gene transcript level of p23 and viral coat protein separately. Virus replication and its coat protein expression were detected by fluorescent in situ hybridization and Western blot, respectively. The effect of p23 was further confirmed by using artificial microRNA inoculation-mediated silencing. Results showed that the two mutants were able to replicate in protoplasts but unable to move from inoculated leaves to newly emerged leaves. Both the p23 and the CP genes of HCRSV were detected in the newly emerged leaves of infected plants but CP was not detected by Western blot and no symptom was observed on those leaves at 19 days post inoculation. This study demonstrates that when p23 is prevented from entering the nucleus, it results in restriction of virus long distance movement which in turn abrogates symptom expression in the newly emerged leaves. We conclude that the p23 protein of HCRSV is required for virus long distance movement.
p23 是 Hibiscus chlorotic ringspot virus（木槿环斑病毒）中一种独特的蛋白，属于 Tombusviridae 科 Carmovirus 属。我们之前的研究结果表明，p23 对宿主特异性复制是不可或缺的，并且定位于细胞核内，具有一个新的核定位信号。为了研究 p23 的其他功能，我们引入了导致碱性氨基酸赖氨酸 (K)、精氨酸 (R) 和组氨酸 (H) 缺失的突变，这些突变会破坏其核定位，用于测试其对病毒复制和体内病毒运动的影响。我们分别通过引物特异性反转录 PCR 检测 p23 和病毒外壳蛋白的基因转录水平。通过荧光原位杂交和 Western blot 检测病毒复制和外壳蛋白表达。通过人工 microRNA 接种介导的沉默进一步证实了 p23 的作用。结果表明，这两个突变体能够在原生质体中复制，但不能从接种叶片转移到新出现的叶片。感染植物的新出现叶片中均检测到了 p23 和 HCRSV 的 CP 基因，但 Western blot 未检测到 CP，在接种后 19 天，这些叶片上没有观察到症状。这项研究表明，当 p23 被阻止进入细胞核时，会限制病毒的长距离运动，从而导致新出现的叶片上症状表达的缺失。我们得出结论，HCRSV 的 p23 蛋白是病毒长距离运动所必需的。
