Characterization of the K1 Protein of Kaposi Sarcoma-associated Herpesvirus and Development of a Novel Vaccine for Protection against Rhadinovirus Infection

Rhesus monkey rhadinovirus (RRV) is a γ-herpesvirus that is closely related tohuman Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relativeto KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo modelsystem for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHVand RRV plays key roles in the establishment and maintenance of these herpesviruses. Wehave constructed a RRV recombinant virus (RRVΔLANA/GFP) in which the RRV LANAopen-reading frame has been disrupted with a GFP expression cassette generated byhomologous recombination. Integrity of the recombinant virus was confirmed by diagnosticPCR, restriction digest, Southern blot analysis, and whole-genome sequencing. We comparedsingle-step and multi-step replication kinetics of RRVΔLANA/GFP, RRV-GFP, wild-type(WT) RRV H26-95, and a revertant virus using traditional plaque assays as well as real-timeQPCR based genome quantification assays. The RRVΔLANA/GFP recombinant virusexhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV,or the revertant virus. This was observed upon de novo infection and in the absence ofchemical inducers such as phorbol esters. In addition, by employing a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRVΔLANA/GFP virus displayed increased lyticgene transcription at all timepoints post-infection compared to RRV-GFP. Moreover, wehave also examined several cellular genes that are known to be repressed by KSHV LANAand report that these genes are de-repressed during de novo lytic infection with theRRVΔLANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRVΔLANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-