Rajamäki Minna-Liisa, Kelloniemi Jani, Alminaite Agne, Kekarainen Tuija, Rabenstein Frank, Valkonen Jari P T
Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland.
Virology. 2005 Nov 10;342(1):88-101. doi: 10.1016/j.virol.2005.07.019. Epub 2005 Aug 19.
The P1 cistron encodes the first and most variable part of the polyprotein of potyviruses. A site tolerant to a pentapeptide insertion at the N-terminus of Potato virus A P1 (Genome Res. 12, 584-594) was used to express heterologous proteins (insertions up to 783 nucleotides) with or without flanking new proteolytic sites. Aequorea victoria green fluorescent protein (GFP) accumulated to high levels when proteolytically released from P1 and showed strong fluorescence in leaves systemically infected with vector virus. Deletions in GFP and adjacent viral sequences emerged 2-4 weeks after infection, revealing putative recombination hot spots. The inserts in P1 diminished infectivity host-specifically, reduced virus accumulation in protoplasts and systemically infected leaves, alleviated symptoms and reduced accumulation of mRNA and HCpro in cis in a virus-free system. This heterologous protein expression site is the first within a protein-encoding cistron and the third in the genome of potyviruses.
P1顺反子编码马铃薯Y病毒多聚蛋白的首个且最具变异性的部分。利用马铃薯A病毒P1的N端对五肽插入具有耐受性的位点（《基因组研究》12卷，584 - 594页）来表达异源蛋白（插入片段长达783个核苷酸），插入片段带有或不带有侧翼新的蛋白酶切位点。当从P1上经蛋白酶切释放后，维多利亚水母绿色荧光蛋白（GFP）大量积累，并在被载体病毒系统侵染的叶片中发出强烈荧光。感染后2 - 4周，GFP及相邻病毒序列中出现了缺失，揭示出假定的重组热点。P1中的插入片段以宿主特异性方式降低了感染性，减少了原生质体及系统侵染叶片中病毒的积累，减轻了症状，并在无病毒系统中顺式减少了mRNA和HCpro的积累。这个异源蛋白表达位点是编码蛋白的顺反子内的首个此类位点，也是马铃薯Y病毒基因组中的第三个此类位点。
