Satyanarayana Tatineni, Gowda Siddarame, Ayllón María A, Albiach-Martí María R, Rabindran Shailaja, Dawson William O
Citrus Research and Education Center, University of Florida, Lake Alfred, Florida 33850, USA.
J Virol. 2002 Jan;76(2):473-83. doi: 10.1128/jvi.76.2.473-483.2002.
Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3' genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3'-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3' genes.
柑橘衰退病毒（CTV）是长线形病毒科的成员，具有19.3 kb的正链RNA基因组，该基因组被组织成12个开放阅读框（ORF），其中3'端的10个基因通过一组嵌套的9个或10个3'共末端亚基因组mRNA（sgRNA）进行表达。与基因组RNA和亚基因组RNA互补的大量负链RNA在受感染细胞中积累。正如RNA病毒的特征一样，野生型CTV产生的正链比负链多，基因组RNA和亚基因组RNA的正链与负链比例估计分别为10至20:1和40至50:1。然而，一个缺失所有3'端基因的突变体能够高效复制，但产生正链与负链的比例显著降低，为1至2:1。对3'端基因的缺失分析表明，p23开放阅读框参与了RNA的不对称积累。一个在第五个密码子后导致移码的突变导致RNA积累几乎对称，这表明是p23蛋白而非p23开放阅读框内的顺式作用元件控制着CTV RNA的不对称积累。p23开放阅读框中进一步的框内缺失突变表明，包含RNA结合域和锌指结构域的46至180位氨基酸残基对于不对称RNA积累是不可或缺的，而N端的5至45位和C端的181至209位氨基酸残基并非绝对必需。锌指结构域中保守的半胱氨酸残基突变为丙氨酸导致p23蛋白失活，表明锌指结构参与了不对称RNA积累。p23基因功能的缺失表现为负链RNA积累大幅增加，而正链RNA仅适度减少。此外，在功能性p23基因存在的情况下，负链外壳蛋白（CP）亚基因组RNA积累的大幅减少导致CP基因的表达增加了12至15倍。显然，过量的负链亚基因组RNA降低了相应正链亚基因组RNA作为信使的可用性。因此，p23蛋白通过下调负链RNA积累来控制CTV RNA的不对称积累，并间接增加3'端基因的表达。
