Function of Rta is essential for lytic replication of murine gammaherpesvirus 68

Rta, encoded primarily by open reading frame 50, is well conserved among ga mmaherpesviruses. It has been shown that the Rta proteins of Epstein Barr v irus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8), and m urine 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 gammaher pesviruses. Since there are cell lines that can support efficient productiv e 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 i nfected cells. Rta, functioning as a transcriptional activator, can activat e the viral promoter of early lytic genes. The amino acid sequence alignmen ts 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 traps-activation domain at the C terminus. We constructe d two mutants of MHV-68 Rta, Rd1 and Rd2, with deletions of 112 and 243 ami no acids from the C terminus, respectively. Rd1 and Rd2 could no longer tra ps-activate the promoter of MHV-68 gene 57, consistent with the deletions o f their traps-activation domains at the C terminus. Furthermore, Rd1 and Rd 2 were able to function as dominant-negative mutants, inhibiting traps-acti vation of wild-type Rta. To study whether Rd1 and Rd2 blocked viral lytic r eplication, purified virion DNA was cotransfected with Rd1 or Rd2 into fibr oblasts. Expression of viral lytic proteins was greatly suppressed, and the yield of infectious viruses was reduced up to 10(4)-fold. Stable cell line s 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 ab ility of Rta dominant-negative mutants to inhibit productive infection sugg ests that the traps-activation function of Rta is essential for MHV-68 lyti c replication. We propose that a single viral protein, Rta, governs the ini tiation of MHV-68 lytic replication during both reactivation and productive de novo infection.