Gammaherpesviruses (GHVs) are ubiquitous, large DNA viruses that include Epstein- Barr virus and Kaposi sarcoma-associated herpesvirus (KSHV), which cause lymphomas and other cancers in immunocompromised patients. GHVs encode oncogenic factors that maintain viral latency indefinitely within the host and dysregulate the cell cycle. One of these viral factors is the latency-associated nuclear antigen (LANA), a conserved, DNA-binding protein encoded by gamma-2-herpesviruses, including KSHV and murine gammaherpesvirus-68 (MHV68). LANA fulfills critical roles during latency, including maintenance and replication of the latent viral episome. LANA also performs important functions during lytic replication, which have only been superficially investigated. Thus, we conducted a mass spectrometry-based interaction screen for proteins that engage MHV68 LANA during lytic replication. We identified both viral and cellular proteins, several of which are conserved interactions identified in KSHV LANA proteomic screens, including the high-priority factor Hsc70. mLANA interacts with and enhances Hsc70 nuclear accumulation in lytically-infected cells. Moreover, Hsc70 function is necessary for efficient lytic replication, as its inhibition delays viral protein synthesis and impairs viral DNA replication in infected cells. To better define mlANA functions in latency, we developed a recombinant virus – O73.loxP MHV68 – to enable cell-type and time-specific deletion of mLANA by Cre-mediated recombination in vivo. O73.loxP MHV68 replicated normally, established latency efficiently, and supported reactivation in C57BL/6 mice. However, O73.loxP MHV68 infection of transgenic mice that express Cre in B cells resulted in a drastic reduction of latently-infected B cells, but not peritoneal exudate cells when compared to mice infected with wildtype MHV68. Moreover, induced deletion of mLANA after latency establishment reduced latent infection of splenocytes in O73.loxP-infected mice expressing tamoxifen-inducible Cre, suggesting that mLANA contributes to latency maintenance. Finally, we generated a modified version of O73.loxP MHV68 that encodes fluorescent markers to detect infected, mLANA positive/negative cells in vivo, toward defining mLANA influence on specific subpopulations of cells. These studies expand the knowledge of LANA’s functions in GHV infection by demonstrating that LANA interacts with cellular factors to promote efficient lytic infection and that it is necessary for latency maintenance during in vivo GHV infection.
|Advisor:||Forrest, James C.|
|Commitee:||Boehme, Karl W., Gray, Wayne L., Ponnappan, Usha, Tackett, Alan J., Zhang, Xuming|
|School:||University of Arkansas for Medical Sciences|
|Department:||Microbiology and Immunology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/09(E), Dissertation Abstracts International|
|Keywords:||Cre-lox, Gammaherpesvirus, Hsc70, Lana, Latency, Lytic|
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