| Marek's disease (MD) is a neurological and lymphoproliferative disorder of chickens caused by a unique, acute-transforming alphaherpesvirus, Marek's disease virus (MDV). During lytic infection and reactivation from latency, MDV expresses a set of phosphoproteins (pp24/pp38) of enigmatic function. The pp38 gene and two additional open reading frames (ORF), R-LORF13a and LORF12, with yet uncharacterized functions, are encoded by a previously described 1.8kb RNA transcript. We have termed LORF12 and R-LORF13a as Mys2 ( Mystery 2) and HEP (BamHI-H Encoded Protein), respectively. To better understand the transcriptional expression of pp38, HEP, and LORF12, we performed RT-PCR and 3' RACE to precisely map the products expressed in this region. We found that not only is the previously described 1.8kb RNA transcribed, but three additional transcripts are expressed within this region as well, capable of encoding pp38, HEP, and an LORF12-specific transcript. To examine pp38 function during MDV infection, we initially constructed two recombinants designated as RB1Bpp38/eGFP and RB1Bpp38/smGFP and have been renamed to RB1Bpp38/eGFP-LORF12 and RB1Bpp38/smGFP+LORF12, respectively. The major structural differences between these viruses is the variant of GFP expressed, direct (RB1Bpp38/eGFP-LORF12) or indirect (RB1Bpp38/smGFP+LORF12) fusion of GFP to pp38, and the absence (RB1Bpp38/eGFP-LORF12) or presence of the full-length LORF12 gene (RB1Bpp38/smGFP+LORF12). RB1Bpp38/eGFP-LORF12 was highly attenuated, while RB1Bpp38/smGFP+LORF12 exhibited pathogenesis similar to parent virus and retained oncogenicity. To explain the difference in pathogenicity, we constructed RB1Bpp38/eGFP+LORF12 and RB1Bpp38/smGFP-LORF12. These viruses are structurally similar to RB1Bpp3 8/eGFP-LORF 12 and RB1Bpp38/smGFP+LORF12, other than encoding LORF12 (RB1Bpp38/eGFP+LORF12) or having the amino terminal portion of LORF12 gene deleted (RB1Bpp38/smGFP-LORF12). In vivo the RB1Bpp38/smGFP+LORF12 replicated to a greater titer than RB1Bpp38/smGFP-LORF12 in both splenocytes and PBLs. In contrast to this, the RB1Bpp38/eGFP recombinants were attenuated, although considerably less so by RB1Bpp38/eGFP+LORF12. These results suggest anon-essential function for LORF12 relative to MDV infection and that expression of eGFP may have interfered with ability of RB1Bpp38/eGFP-LORF12 to replicate in vivo. Using fluorescence resonance energy transfer (FRET), we have demonstrated that HEP interacts with the splicing factor complex protein, SmB, suggesting that HEP may block splicing of the full-length 1.8kb pp38 RNA transcript, favoring lytic infection and reactivation from latency. |
