Wu T T, Tong L, Rickabaugh T, Speck S, Sun R
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.
J Virol. 2001 Oct;75(19):9262-73. doi: 10.1128/JVI.75.19.9262-9273.2001.
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 gamma HV68) 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 their trans-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 10(4)-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.
Rta主要由开放阅读框50编码，在γ疱疹病毒中高度保守。研究表明，爱泼斯坦-巴尔病毒（EBV）、卡波西肉瘤相关疱疹病毒（KSHV，即HHV-8）和鼠γ疱疹病毒68（MHV-68，也称为γHV68）的Rta蛋白在病毒从潜伏状态重新激活过程中发挥重要作用。然而，Rta在γ疱疹病毒有 productive de novo感染期间的作用尚未明确。由于存在能够支持MHV-68高效 productive de novo感染但不支持EBV或KSHV感染的细胞系，我们研究了MHV-68 Rta在 productive感染细胞中启动病毒裂解复制是否发挥作用。Rta作为转录激活因子，可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，其功能域的组织方式相似，N端为DNA结合和二聚化结构域，C端为反式激活结构域。我们构建了两个MHV-68 Rta突变体，Rd1和Rd2，分别从C端缺失了112和243个氨基酸。Rd1和Rd2不再能够反式激活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一致。此外，Rd1和Rd2能够作为显性负突变体发挥作用，抑制野生型Rta的反式激活。为了研究Rd1和Rd2是否阻断病毒裂解复制，将纯化的病毒体DNA与Rd1或Rd2共转染到成纤维细胞中。病毒裂解蛋白的表达受到极大抑制，感染性病毒的产量降低了高达10^4倍。建立了组成性表达Rd2的稳定细胞系并感染MHV-68。在这些细胞系中，病毒的立即早期基因rta和早期基因tk的转录减少。Rd2的存在还导致病毒裂解蛋白表达和病毒体产生减弱。Rta显性负突变体抑制 productive感染的能力表明，Rta的反式激活功能对于MHV-68裂解复制至关重要。我们提出，单一病毒蛋白Rta在重新激活和 productive de novo感染期间均控制着MHV-68裂解复制的起始。
