Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

Abstract

The vaccinia virus E9 protein, the catalytic subunit of the DNA polymerase holoenzyme, is inherently distributive under physiological conditions, although infected cells contain a highly processive form of the enzyme. The viral A20 protein was previously characterized as a stoichiometric component of the processivity factor, and an interaction between A20 and E9 was documented in vivo. A20 has been shown to interact with D4, the virally encoded uracil DNA glycosylase (UDG), by yeast-two hybrid and in vitro analysis. Here we confirm that UDG and A20 interact in vivo and show that temperature-sensitive viruses with lesions in the D4R gene show a profound defect in DNA synthesis at the non-permissive temperature. Moreover, cytoplasmic extracts prepared from these infections lack processive polymerase activity in vitro, implicating D4 in the assembly or activity of the processive polymerase. Upon overexpression of 3؋FLAG-UDG, A20, and E9 in various combinations, we purified dimeric and trimeric UDG-A20 and UDG-A20-polymerase complexes, respectively. These complexes are stable in 750 mM NaCl and can be further purified by Mono Q chromatography. Notably, the trimeric complex displays robust processive polymerase activity, and the dimeric complex can confer processivity on purified E9. Consistent with previous reports that the catalytic activity of UDG is dispensable for virus replication in tissue culture, we find that the role of UDG role in the polymerase complex is not diminished by mutations targeting residues involved in uracil recognition or excision. Our cumulative data support the conclusion that A20 and UDG form a heterodimeric processivity factor that associates with E9 to comprise the processive polymerase holoenzyme.Vaccinia virus, the prototypic orthopoxvirus, shows a significant degree of genetic autonomy from the host cell. Because all stages of the viral life cycle take place in the cytoplasm, vaccinia encodes most, if not all, of the factors involved in the replication of its 192-kb genome. The repertoire of essential replication functions appears to include: E9 (replicative DNA polymerase), A20 (stoichiometric component of the processivity factor), D5 (DNA-independent dNTPase), B1 (Ser/Thr protein kinase), I3 (single strand DNA-binding protein), A22 (Holliday junction resolvase), and D4 (uracil DNA glycosylase, UDG) 2 (Ref. 1, reviewed in Refs. 2 and 3). Other proteins implicated in the process of genome replication or maintenance include A50 (DNA ligase), H6 (topoisomerase), F2 (dUTPase), J2 (thymidine kinase), A48 (thymidylate kinase), and F4/I4 (ribonucleotide reductase) (reviewed in Refs. 2 and 3).In most cases, a processive DNA polymerase comprises the core of the replication machinery. Processivity, which enables polymerases to replicate long templates rapidly and accurately, is not an intrinsic property of most polymerases but rather is conferred by accessory proteins. Indeed, the vaccinia virus E9 protein, which is the catalytic subunit of the polymerase, is inherently distributive under phys...

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

“…6E). Previous studies have shown that UNGs from human, HCMV, and vaccinia virus interact with PCNA or its viral homolog and colocalize to the DNA replication foci (50,53,67). Moreover, the interaction of UNG with the processivity factor of vaccinia virus increased the processivity of the viral DNA polymerase (50,53,67).…”

“…Previous studies have shown that UNGs from human, HCMV, and vaccinia virus interact with PCNA or its viral homolog and colocalize to the DNA replication foci (50,53,67). Moreover, the interaction of UNG with the processivity factor of vaccinia virus increased the processivity of the viral DNA polymerase (50,53,67). It is thus possible that cellular UNG2 activity may be sufficient at the early stage of viral DNA replication, whereas the viral enzyme is important in a later stage of viral DNA replication through interaction with viral replication factors, thereby enhancing the efficiency of viral replication.…”

“…The vaccinia virus D4R gene, which encodes the viral UNG, is essential for replication in tissue culture, although the catalytic activity is dispensable (14), suggesting that vaccinia virus UNG may participate in the formation of multiprotein DNA replication complexes. Indeed, the interaction of vaccinia virus UNG with A20 (a stoichiometric component of the viral processivity factor), along with E9 (viral DNA polymerase), is necessary and sufficient for the processive polymerase holoenzyme (67). The UNG encoded by HCMV UL114 also was shown to associate with ppUL44 (viral DNA polymerase processivity factor), and UL114 functions as part of the viral DNA replication complex to increase the efficiency of both early-and late-phase viral DNA synthesis (53).…”

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

“…Curiously, this function of UNG is independent of its catalytic activity. Similarly, vaccinia virus UNG protein, but not its catalytic activity, is essential for the viral replicative cycle by the formation of a large complex to support processive DNA synthesis (23,24). UNG is also known to be recruited to DSB foci in association with γH2AX (25).…”

Poxvirus DNA primase

“…The essential role of D4 in DNA replication (6) is independent of its uracil DNA glycosylase activity (7), which presumably has a facultative repair function. The A20 and D4 proteins physically interact (8,9) and together provide processivity for the DNA polymerase (10). The B1 kinase phosphorylates a cellular DNA-binding protein called BAF and prevents the latter from blocking VACV DNA replication (11).…”