The “Bridge” in the Epstein-Barr Virus Alkaline Exonuclease Protein BGLF5 Contributes to Shutoff Activity during Productive Infection

Abstract

Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible “bridge” that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.

Epstein-Barr Virus-Specific Humoral Immune Responses in Health and Disease

“…Separately, it was demonstrated that antibodies in sera of NPC patients could neutralise a viral alkaline exonuclease (DNAse) function which had diagnostic relevance, but only when using native DNAse enzyme prepared from P3HR1 cells (Cheng et al 1980;Stolzenberg et al 1996;Middeldorp and Ooka unpublished data). This proved to be due to small amino acid variations between type 1 (B95-8 prototype) and type 2 (AG786, P3HR1 prototype) EBV strains altering the antigenic structure of the EBV DNAse, but not its function (Liu et al 1998;Paramita et al 2007;Horst et al 2012). In 1983, Glaser et al, described first evidence that EA(R) was encoded by a distinct gene compared to EA(D), which subsequently is defined as the BHRF1 open reading frame, encoding the 17 KDa viral Bcl-2 homologue.…”

Mitochondrial Nucleases ENDOG and EXOG Participate in Mitochondrial DNA Depletion Initiated by Herpes Simplex Virus 1 UL12.5

“…Both mutations abolish the ability of UL12 to complement the growth defect of a UL12-null mutant virus (29). Mutagenesis of EBV, HCMV, and KSHV UL12 orthologs has also demonstrated that residues analogous to UL12 G336, S338, and D340 are critical for nuclease activity (30,(32)(33)(34). We included three additional mutations that have also been shown to eliminate detectable nuclease activity in vitro and abolish complementing activity in vivo; ⌬N, L150K, and ⌬C (23).…”

Immune Evasion by Epstein-Barr Virus

“…This process, termed shutoff, is mediated by the EBV DNase (alkaline exonuclease) BGLF5, which is expressed with early kinetics during the productive phase of infection (Zuo et al 2008). BGLF5's additional RNase function utilizes the same catalytic site as its DNase activity, yet the substrate-binding site appears only partly shared by DNA and RNA substrates (Horst et al 2012). The promiscuous RNA degradation induced by EBV BGLF5 can affect immunologically relevant proteins, including TLR2 and TLR9 that are capable of sensing EBV infection (Gaudreault et al 2007;van Gent et al 2011van Gent et al , 2015.…”