Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

Abstract

Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (⌬UNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68⌬UNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCEHerpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the initial site of acute replication was associated with a substantial delay of latency establishment in the spleen. The levels of host UNG were substantially lower in the lung compared to the spleen, suggesting that herpesviruses encode a viral UNG to compensate for reduced host enzyme levels in some cell types and tissues. These data suggest that intervention at the site of initial replicative expansion can delay the establishment of latency, a hallmark of chronic herpesvirus infection.A ll herpesvirus genomes encode a homolog of the host uracil DNA glycosylase. If not repaired, uracil misincorporation during DNA replication (1, 2) or conversion upon cytosine deamination (2, 3) will lead to a C:G-to-T:A transition mutation that may have severe consequences for genomic integrity (1). In the host, the recognition and removal of uracils is mediated by members of the uracil DNA glycosylase (UDG) superfamily. UNG2 is the predominant nuclear enzyme responsible for initiating repair (2-6). The role of a seemingly redundant herpesvirus UNG in viral replication is ...

Functions and Malfunctions of Mammalian DNA-Cytosine Deaminases

“…Furthermore, it has been shown that inactivation of vUNG in several herpes viruses reduces viral replication. 247–249 It would be interesting to investigate whether vUNG proteins provide protection against the action of AID/APOBEC family enzymes on herpes viral genomes.…”

Murine Gammaherpesvirus 68 Pathogenesis Is Independent of Caspase-1 and Caspase-11 in Mice and Impairs Interleukin-1β Production upon Extrinsic Stimulation in Culture

“…Rabbit anti-LANA antibody was kindly provided by Scott Tibbetts. Affinity-purified chicken anti-ORF75c antibody (Gallus Immunotech, Cary, NC) was previously described (32). Detection was performed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG (GE Healthcare, Buckinghamshire, United Kingdom), HRP-conjugated streptavidin IgG (Rockland, Gilbertsville, PA), or HRP-conjugated goat anti-chicken (Gallus Immunotech).…”

5-Ethynyl-2′-deoxycytidine and 5-ethynyl-2′-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses

“…The virus encodes factors to prevents this incorporation by either decreasing the pools of dUTP through the activity of viral dUTPases or through recognition of incorporated deoxyuridine by viral UNG enzymes (62)(63)(64). While many viral dUTPases or vUNG are dispensable in normal cell culture models of infection, these viral enzymes are required for viral replication and pathogenesis for in vivo models of infection (65)(66)(67)(68)(69)(70). Labeling with EdU for longer time pulses, especially when creating labeled infectious virions, are reported to decrease titers in cells incubated with EdU (26,71).…”