Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68
Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68
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DOI：
10.1128/jvi.75.19.9262-9273.2001
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发表时间：
2001-10
影响因子：
5.4
通讯作者：
Tingting Wu;L. Tong;T. Rickabaugh;S. Speck;R. Sun
中科院分区：
文献类型：
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作者：
Tingting Wu;L. Tong;T. Rickabaugh;S. Speck;R. Sun
摘要RTA主要由开放阅读框50编码，在伽马疱疹病毒中高度保守。已有研究表明，EB病毒、卡波西肉瘤相关疱疹病毒和小鼠伽马疱疹病毒68的Rta蛋白在病毒潜伏期重新激活过程中起重要作用。然而，在伽马疱疹病毒中，RTA在新生感染中的作用还没有被表征。由于有一些细胞系可以支持MHV-68有效的从头感染，但不支持EBV或KSHV，我们研究了MHV-68 RTA是否在高效感染细胞中启动病毒裂解复制的作用。RTA作为转录激活剂，可以激活早期裂解基因的病毒启动子。RTA同源物的氨基酸序列比对表明，它们的功能结构域的组织结构相似，N端有DNA结合和二聚化结构域，C端有反式激活结构域。我们构建了MHV-68RTA的两个突变体Rd1和Rd2，C末端分别缺失112和243个氨基酸。Rd1和Rd2不再反式激活MHV-68基因57的启动子，这与它们C端反式激活结构域的缺失是一致的。此外，Rd1和Rd2能够作为显性-负性突变体发挥作用，抑制野生型RTA的反式激活。为了研究Rd1和Rd2是否能阻断病毒的裂解复制，将纯化的病毒DNA与Rd1或Rd2共转染成纤维细胞。病毒裂解蛋白的表达受到了极大的抑制，感染性病毒的产量减少了104倍。建立了稳定表达RD2的细胞系，并感染MHV-68。在这些细胞系中，病毒的即刻早期基因rta和早期基因tk的转录减少。RD2的存在也导致病毒裂解蛋白表达和病毒粒子产生的减弱。RTA显性-负性突变体抑制生产性感染的能力表明，RTA的反式激活功能对于MHV-68裂解复制是必不可少的。我们认为，单一的病毒蛋白，RTA，控制着MHV-68裂解复制的启动，在重新激活和产生从头感染的过程中。
ABSTRACT Rta, encoded primarily by open reading frame 50, is well conserved among gammaherpesviruses. It has been shown that the Rta proteins of Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV-68; also referred to as γHV68) play an important role in viral reactivation from latency. However, the role of Rta during productive de novo infection has not been characterized in gammaherpesviruses. Since there are cell lines that can support efficient productive de novo infection by MHV-68 but not EBV or KSHV, we examined whether MHV-68 Rta plays a role in initiating viral lytic replication in productively infected cells. Rta, functioning as a transcriptional activator, can activate the viral promoter of early lytic genes. The amino acid sequence alignments of the Rta homologues suggest that the organizations of their functional domains are similar, with the DNA binding and dimerization domains at the N terminus and the trans-activation domain at the C terminus. We constructed two mutants of MHV-68 Rta, Rd1 and Rd2, with deletions of 112 and 243 amino acids from the C terminus, respectively. Rd1 and Rd2 could no longer trans-activate the promoter of MHV-68 gene 57, consistent with the deletions of theirtrans-activation domains at the C terminus. Furthermore, Rd1 and Rd2 were able to function as dominant-negative mutants, inhibiting trans-activation of wild-type Rta. To study whether Rd1 and Rd2 blocked viral lytic replication, purified virion DNA was cotransfected with Rd1 or Rd2 into fibroblasts. Expression of viral lytic proteins was greatly suppressed, and the yield of infectious viruses was reduced up to 104-fold. Stable cell lines constitutively expressing Rd2 were established and infected with MHV-68. Transcription of the immediate-early gene, rta, and the early gene, tk, of the virus was reduced in these cell lines. The presence of Rd2 also led to attenuation of viral lytic protein expression and virion production. The ability of Rta dominant-negative mutants to inhibit productive infection suggests that the trans-activation function of Rta is essential for MHV-68 lytic replication. We propose that a single viral protein, Rta, governs the initiation of MHV-68 lytic replication during both reactivation and productive de novo infection.