A Tissue Culture Model of Murine Gammaherpesvirus Replication Reveals Roles for the Viral Cyclin in Both Virus Replication and Egress from Infected Cells

Francine M Scott,Samuel H Speck,David A Leib

doi:10.1371/journal.pone.0093871

Abstract

Passage through the eukaryotic cell cycle is regulated by the activity of cyclins and their cyclin-dependent kinase partners. Rhadinoviruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), encode a viral homologue of mammalian D-type cyclins. In MHV68, the interaction of the viral cyclin with its CDK partners is important for acute replication in the lungs following low dose inoculation. Attempts to further study this requirement in vitro have been limited by the lack of available tissue culture models that mimic the growth defect observed in vivo. It is hypothesized that analysis of virus replication in a cell line that displays properties of primary airway epithelium, such as the ability to polarize, might provide a suitable environment to characterize the role of the v-cyclin in virus replication. We report here MHV68 replication in the rat lung cell line RL-65, a non-transformed polarizable epithelial cell line. These analyses reveal a role for the v-cyclin in both virus replication, as well as virus egress from infected cells. As observed for acute replication in vivo, efficient replication in RL-65 cells requires CDK binding. However, we show that the KSHV v-cyclin (K-cyclin), which utilizes different CDK partners (CDK4 and CDK6) than the MHV68 v-cyclin (CDK2 and CDC2), can partially rescue the replication defect observed with a v-cyclin null mutant – both in vitro and in vivo. Finally, we show that MHV68 is shed from both the apical and basolateral surfaces of polarized RL-65 cells. In summary, the RL-65 cell line provides an attractive in vitro model that mimics critical aspects of MHV68 replication in the lungs.

Highlights

The eukaryotic cell cycle is a tightly regulated and sequentially executed pathway by which DNA is duplicated and partitioned into daughter cells
After screening a number of fibroblast and epithelial cell lines in which we failed to identify a significant replication defect of the v-cyclin null virus, we report here murine gammaherpesvirus 68 (MHV68) growth in the rat lung cell line RL-65
RL-65 cells are a spontaneously immortalized, non-transformed epithelial cell line that was originally derived from neonatal rat lungs by careful manipulation of microenvironment to select for a cell type that maintained highly differentiated features in vitro [24]

Summary

Introduction

The eukaryotic cell cycle is a tightly regulated and sequentially executed pathway by which DNA is duplicated and partitioned into daughter cells. Cyclins and their cyclin-dependant kinases (CDKs) form active complexes that facilitate this progression. It is upon mitogenic stimulation that many of these cells upregulate cyclin D which partners with CDK4 and/or 6 to drive entry into the G1 phase of the cell cycle. Cyclin A partners with CDK2 and will promote DNA replication and progression through S phase, as well as entry into the G2 phase of the cell cycle. Eukaryotic cells employ mechanisms to regulate cyclin:CDK activities. Catalytic subunit inhibition and cyclin:CDK assembly promotion by CKIs are just two examples of how the cell cycle can be kept in check [2]

Methods

Results

Conclusion