Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 RTA reactivates murine gammaherpesvirus 68 from latency

Abstract

Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.

FIG. 1.

Activation of MHV-68, EBV, and HHV-8 promoters by homologous RTA proteins. A reporter construct consisting of either the MHV-68 ORF50 promoter (pMRP1.2kb) (A), the MHV-68 ORF57/Mta promoter (p57luc) (B), the EBV BHLF1 promoter (C), or the HHV-8 PAN promoter (pPAN-69luc) (D) was cotransfected with each construct or pFLAG-CMV in the amounts indicated. All transfections were done in BHK-21 or 293T cells. Cell lysates were harvested at 36 h posttransfection and assayed for luciferase activity.

FIG. 2.

Transfection of homologous RTA proteins into MHV-68-latently infected cells. (A) MHV-68-latently infected S11-E cells were electroporated with pFLAG-CMV (V), pFLAG-M/RTA (M), pFLAG-H/RTA (H), or pFLAG-E/RTA (E). Protein from 10⁴ MHV-68-infected BHK-21 cells (+) was included as a positive control, and S is the protein marker standard (Bio-Rad). Western analysis was performed with rabbit hyperimmune serum against MHV-68-infected rabbit cell lysates and a murine monoclonal actin antibody as a control for protein loading. (B) MHV-68-latently infected S11-E cells were elec-troporated with of pFLAG (V), pFLAG-M/RTA (M), pFLAG-H/RTA (H), or pFLAG-E/RTA (E). Total DNA from transfected cells was harvested at the indicated time points posttransfection. The DNA was digested with HindIII and subjected to Southern analysis with a probe derived from a 0.8-kb region adjacent to the terminal repeat region of MHV-68. (C) Supernatants from the transfected S11E cells were collected at the time points indicated, and the viral titer was measured by plaque assay.

FIG. 3.

Construction of chimeric RTA proteins and their activation of gammaherpesvirus promoters. (A) The DBD and activation domain (AD) are indicated for the three RTA proteins. The activation domains of the proteins were swapped as indicated in the diagram, and the amino acid sequences that were used are labeled. (B) 293T cells were transfected with pFLAG (V), pFLAG-H/RTA (H), pFLAG-M/RTA (M), pFLAG-E/RTA (E), pFLAG-M-H/RTA (M-H), pFLAG-H-M/RTA (H-M), pFLAG-E-M/RTA (E-M), or pFLAG-E-H/RTA (E-H). Cell lysates were harvested 24 h posttransfection, and a Western blot was performed, using a monoclonal antibody against FLAG (Sigma). The lane marked S is the protein marker standard (Bio-Rad). A reporter construct consisting of either the HHV-8 Pan promoter (E), the EBV BHLF1 promoter (D), or the MHV-68 ORF57 promoter (C was cotransfected with each construct or pFLAG-CMV in the amounts indicated into 293T cells. Cell lysates were harvested at 36 h posttransfection and assayed for luciferase activity.

FIG. 4.

Transfection of chimeric RTA proteins into MHV-68-latently infected cells. (A) MHV-68-latently infected S11-E cells were electroporated with pFLAG (V), pFLAG-H/RTA (H), pFLAG-M/RTA (M), pFLAG-E/RTA (E), pFLAG-M-H/RTA (M/H), pFLAG-H-M/RTA (H/M), pFLAG-E-M/RTA (E/M), or pFLAG-E-H/RTA (E/H). Protein from 10⁴ MHV-68-infected BHK-21 cells (+) was included as a positive control, and lane S is the protein marker standard (Bio-Rad). Western analysis was performed with rabbit hyperimmune serum against MHV-68-infected rabbit cell lysates. (B) Supernatants from the transfected S11E cells were collected at the time points indicated, and the viral titer was measured by plaque assay. The percentage of reactivation was determined by comparing the amount of virions produced by each construct to the amount of virions produced by electroporation of pFLAG-M/RTA, which was set at 100%.

FIG. 5.

Cotransfection of a recombinant MHV-68 RTA-deficient virus with each of the RTA constructs. (A) A BAC clone containing a recombinant MHV-68 RTA-deficient virus was cotransfected with pFLAG (V), pFLAG-H/RTA (H), pFLAG-M/RTA (M), pFLAG-E/RTA (E), pFLAG-M-H/RTA (M-H), pFLAG-H-M/RTA (H-M), pFLAG-E-M/RTA (E-M), or pFLAG-E-H/RTA (E-H) into BHK-21 (shown) or 293T cells. Six days posttransfection, DNA was extracted from these cells and real-time PCR was performed with M9 primers and probe. (B) An MHV-68 ORF50 promoter construct (pMRP1.2kb) was cotransfected with each construct or pFLAG-CMV in the amounts indicated. All transfections were done with 293T cells. Cell lysates were harvested at 36 h posttransfection and assayed for luciferase activity.