Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong chronic infections characterized by initial productive replication (lytic phase) followed by persistence of latent viral episomes, which places the host at risk of developing lymphoproliferative diseases and other malignancies. This work discusses the roles of GHV immediate-early genes in pathogenesis that we examined by developing two separate viral genetic strategies.
Lytic viral replication is necessary for murine gammaherpesvirus 68 (MHV68) to disseminate to distal latency reservoirs. Dogma suggests homeostatic reactivation of GHVs maintains latency, but roles for acute replication and periodic reactivation from latency in long-term GHV maintenance are unclear. To address this question, we developed a viral genetic strategy that enables cre-mediated conditional ablation of essential MHV68 lytic genes from B cells. Despite complete deletion of the genes essential for lytic replication, these mutant viruses established WT levels or latency, but exhibited a severe defect in reactivation from latency. Additionally, in the complete absence of viral reactivation, polyclonal expansion of latently infected B cells was sufficient to increase latency. These data demonstrate that the B cell compartment is important to viral reactivation, but productive viral replication in B cells and periodic homeostatic reactivation are not necessary for MHV68 latency establishment or maintenance.
The human GHV Kaposi sarcoma-associated herpesvirus (KSHV), is phenotypically latent in infected cells, unlike MHV68 which undergoes productive replication in infected cells and produces infectious particles. We sought to determine if KSHV and MHV68 latency-associated nuclear antigen (LANA) homologs are functionally interchangeable, or if the phenotypic differences in KSHV and MHV68 infection could be attributed to the main latency antigen. A kLANA knock-in (KLKI) MHV68 expressed that expressed kLANA in place of mLANA was replication competent both in vitro and in vivo. However, KLKI MHV68 exhibited slower growth kinetics and reduced output titers compared to WT MHV68. Following intranasal inoculation of mice KLKI MHV68 established and maintained latency in splenocytes but did not reactivate efficiently. kLANA repressed the MHV68 promoter for the major lytic transactivator (RTA), while mLANA did not. Further, provision of MHV68 RTA in-trans rescued KLKI MHV68 replication in culture. Our findings suggest that kLANA repression of lytic replication, which was previously shown for KSHV, is transferable to MHV68, and is a plausible explanation for the latent phenotype exhibited by KSHV.
These findings reveal the roles of three separate immediate early genes in all aspects of GHV infections- replication, dissemination, latency, reactivation and long-term genome carriage. Building on this work, we hope to be able to develop more nuanced studies to decipher GHV pathogenesis in vivo.
|Advisor:||Forrest, James C.|
|Commitee:||Boehme, Karl W., Cannon, Martin J., Kelly, Thomas, Zhang, Xuming|
|School:||University of Arkansas for Medical Sciences|
|Department:||Microbiology and Immunology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 81/2(E), Dissertation Abstracts International|
|Subjects:||Virology, Microbiology, Immunology|
|Keywords:||Dissemination, Gammaherpesvirus, Latency, MHV68, Reactivation|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be