Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Recruits Uracil DNA Glycosylase 2 at the Terminal Repeats and Is Important for Latent Persistence of the Virus

Self Cite

The DNA damage response (DDR) of host cells is utilized by a number of viruses to establish and propagate their genomes in the infected cells. We examined the expression of the DDR genes during Kaposi's sarcoma-associated herpesvirus (KSHV) infection of human peripheral blood mononuclear cells (PBMCs). The genes were mostly downregulated, except H2AX, which was upregulated during infection. H2AX is important for gammaherpesvirus infectivity, and its phosphorylation at serine 139 is crucial for maintenance of latency during mouse gamma-herpesvirus 68 (MHV-68) infection. We now also observed phosphorylation of H2AX at serine 139 during KSHV infection. H2AX is a histone H2A isoform shown to interact with the latency-associated nuclear antigen (LANA) encoded by KSHV. Here, we show that LANA directly interacted with H2AX through domains at both its N and C termini. The phosphorylated form of H2AX (␥H2AX) was shown to colocalize with LANA. Chromatin immunoprecipitation (ChIP) assays showed that a reduction in H2AX levels resulted in reduced binding of LANA with KSHV terminal repeats (TRs). Binding preferences of H2AX and ␥H2AX along the KSHV episome were examined by whole-episome ChIP analysis. We showed that ␥H2AX had a higher relative binding activity along the TR regions than that of the long unique region (LUR), which highlighted the importance

Section: Discussionmentioning

confidence: 78%

“…Chromatin immunoprecipitation (ChIP) was carried out as has been described earlier (24,30). Briefly, after the cells were fixed, nuclei were isolated and sonicated in the nuclear lysis buffer to an average DNA length of 700 bp, as confirmed by agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

H2AX Phosphorylation Is Important for LANA-Mediated Kaposi's Sarcoma-Associated Herpesvirus Episome Persistence

Jha

Upadhyay

et al. 2013

Self Cite

“…However, HSV-1 reactivation was measured post-explant from trigeminal ganglia: a tissue composed of terminally differentiated adult neurons with low levels of host UNG (18,36). Reactivation is also reduced in human gammaherpesvirus latency cell systems lacking the EBV viral UNG (BKRF3) alone or in the absence of both the vUNG and the host UNG (13,18,(37)(38)(39). Interestingly, Su et al (13) recently reported that host UNG2 levels decrease as EBV reactivation proceeds, indicating that the viral UNG is needed to promote replication under conditions of low host UNG levels (13).…”

Section: Discussionmentioning

confidence: 99%

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

Minkah

Macaluso

Oldenburg

et al. 2015

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 ...

“…Additionally, recent studies with Kaposi's sarcoma herpesvirus identified human UNG2 as a protein interacting with latencyassociated nuclear antigen. Depletion of UNG2 in Kaposi's sarcoma herpesvirus-positive cells by using siRNA reduced the number of viral genome copies and produced infection-deficient virus (73). Because UNG2 was previously found to interact with several cellular replication proteins, such as PCNA, PRA, and polymerase ␣ (29,50,63), it would be interesting to study whether UNG2 can recruit these cellular machineries to the viral replication compartment for enhancing viral replication efficiency.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

Huang

Wang

et al. 2007

Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediateearly transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.