Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection

Abstract

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.

Deletion of Murine Gammaherpesvirus GeneM2in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

“…To better define roles for the MHV68 latency gene product M2 within distinct populations of B lymphocytes, we generated a recombinant virus to enable cell-type-specific deletion of the M2 gene in infected cells that express Cre recombinase. We previously demonstrated the feasibility and utility of this approach in a study that defined B cell-specific requirements for ORF73 , the gene that encodes the MHV68 latency-associated nuclear antigen 105 (mLANA) (30).…”

“…NIH 3T12 (ATCC CCL-164), 3T12 Flp+ (30), BHK21 (ATCC CCL-10), Vero (ATCC CCL-81), Vero-Cre (47), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamine (cDMEM). Cells were maintained at 37°C in 5% CO 2 and ∼99% humidity.…”

“…Murine embryonic fibroblasts (MEFs) were harvested from C57BL/6 mouse embryos as previously described (34). Previously described viruses used in this study include FRT BAC MHV68 (WT MHV68) (30) and M2.null MHV68 (M2.STOP) (27).…”

“…To generate the M2.loxP BAC, loxP sites were inserted adjacent to the 5’ and 3’ ends of M2 in a FRT BAC template by two successive rounds of en passant mutagenesis as previously described (30) utilizing primers:…”

“…Since GC B cells are critical early targets for GHV infection, determining how specific viral gene products function within these cells to permit latency is fundamental to understanding GHV pathogenesis. We previously reported development of a viral genetic platform that allows for dissection of cell-type-specific roles of viral gene products in vivo (30). Building from this technology, we engineered a recombinant MHV68 in which the gene encoding M2 was flanked by loxP sequences (floxed; M2.loxP) to enable the conditional deletion of M2 in cells that express Cre recombinase.…”