Demonstration that orf2 encodes the feline immunodeficiency virus transactivating (Tat) protein and characterization of a unique gene product with partial Rev activity
Demonstration that orf2 encodes the feline immunodeficiency virus transactivating (Tat) protein and characterization of a unique gene product with partial Rev activity
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DOI：
10.1128/jvi.73.1.608-617.1999
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发表时间：
1999-01-01
影响因子：
5.4
通讯作者：
Elder, JH
中科院分区：
文献类型：
--
作者：
de Parseval, A;Elder, JH
使用长 PCR 技术来扩增猫免疫缺陷病毒 (FIV) 感染细胞中产生的三种大小类别的病毒 mRNA，我们在 env 区域中鉴定了一个新的剪接受体位点，该位点生成两个转录本，每个转录本编码一个 11-kDa 蛋白质 p11(rev)，其功能未知。小尺寸的 mRNA 包括两个双顺反子 orf2/rev mRNA 和两个 rev 样 mRNA，仅由 rev 的第二个外显子组成，编码 15 kDa 蛋白 p15(rev)。 p15(rev) 包含 Rev 的最小效应器结构域，足以介导部分 Rev 活性。双顺反子 mRNA 编码两种不同的蛋白质，一种是与 Rev 相对应的 23 kDa 蛋白质，另一种是由 orf2 基因编码的 9-kDa 蛋白质。 orf2基因产物是一种由79个氨基酸组成的蛋白质，其特征与有蹄类慢病毒的Tat（反式激活蛋白）蛋白质相似。使用 FIV 长末端重复序列 (LTR) 驱动氯霉素乙酰转移酶细菌基因转录的瞬时表达测定表明，orf2 基因反式激活基因表达平均比基础水平高 14 至 20 倍。生成 FIV LTR 的缺失突变体以定位对 orf2 基因介导的反式激活有反应的序列。去除 AP1 位点的 5' 缺失突变体导致 orf2 残留低水平反式激活。使用具有内部缺失的 LTR 突变体的进一步实验确定了相对于帽位点而言位于位置 -126 和 -47 之间的三个区域，这对于时代定向反式激活很重要。这些区域包括 AP1 位点、C/EBP 串联重复序列和 ATF 位点。
The long PCR technique was used to amplify the three size classes of viral mRNAs produced in cells infected by feline immunodeficiency virus (FIV), We identified in the env region a new splice acceptor site that generated two transcripts, each coding for an 11-kDa protein, p11(rev), whose function is unknown. The small-size class of mRNAs included two bicistronic orf2/rev mRNAs and two rev-like mRNAs, consisting only of the second exon of rev and coding for a 15-kDa protein, p15(rev). p15(rev) contained the minimal effector domain of Rev and was sufficient to mediate partial Rev activity. The bicistronic mRNAs encoded two distinct proteins, one of 23 kDa corresponding to Rev and a 9-kDa protein encoded by the orf2 gene. The orf2 gene product is a protein of 79 amino acids with characteristics similar to those of the Tat (transactivator) proteins of the ungulate lentiviruses. Transient expression assays, using the FIV long terminal repeat (LTR) to drive transcription of the bacterial gene for chloramphenicol acetyltransferase demonstrated that the orf2 gene transactivates gene expression an average of 14- to 20-fold above the basal level. Deletion mutants of the FIV LTR were generated to locate sequences responsive to transactivation mediated by the orf2 gene. A 5' deletion mutant that removed the AP1 site resulted in residual low-level transactivation by orf2, Further experiments using LTR mutants with internal deletions identified three regions located between positions -126 and -47 relative to the cap site that were important for era-directed transactivation, These regions include the AP1 site, a C/EBP tandem repeat, and an ATF site.