The A20R Protein Is a Stoichiometric Component of the Processive Form of Vaccinia Virus DNA Polymerase

Abstract

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl 2 or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of ϳ48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent ϳ45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken Vaccinia virus exhibits a high degree of genetic and physical autonomy from the host cell, possessing a genome that encodes more than 200 genes and directing a replicative cycle in which DNA replication, gene expression, and morphogenesis take place solely within the cytoplasm of the infected cell. It is therefore likely that the trans-acting functions required for DNA replication are virally encoded, and several laboratories have engaged in studies aimed at identifying and characterizing these functions. The catalytic DNA polymerase of vaccinia virus is encoded by the E9 gene (6,20,31). The 116-kDa enzyme possesses both polymerase and proofreading exonuclease activity and retains many of the conserved domains that are found within the replicative polymerases of mammalian and yeast cells, as well as those encoded by other large DNA viruses. In addition, the vaccinia virus enzyme, like the catalytic subunit of these other replicative polymerases, is inherently distributive. Our studies on the purified E9 protein indicate that in the presence of 40 mM NaCl or 8 mM MgCl 2 , the enzyme catalyzes the addition of Ͻ10 nucleotides (nt) per primer-template binding event (21). Such distributive behavior would be incompatible with efficient replication of a large genome in vivo, and it is therefore no surprise that a highly processive form of the vaccinia virus enzyme is found in unfractionated cytoplasmic extracts of infected cells (19). This processive enzyme is capable of catalyzing the addition of Ͼ7,000 nt per primer-template binding event.This dichotomy between the dis...

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

“…As shown in Fig. 4B, fUDG co-immunoprecipitates with A20 when both are expressed at endogenous levels (lanes [3][4][5]. This is the first evidence of their interaction in vivo.…”

“…As previously described, the 20-min primer extension reaction was initiated by adding dATP and [ 32 P]TTP after a 3-min preincubation that was performed in the presence of only dCTP and dGTP (5). Reactions were quenched by the addition of an equal volume of 1% SDS-40 mM EDTA and resolved on 0.8% agarose gels containing 0.125 g/ml ethidium bromide.…”

“…2) as appropriate (5,14). Infections were initiated at 37°C and shifted to 31.5°C at 4 hpi to increase the yield of soluble protein.…”

“…Overexpression of fUDG is induced upon co-infection with a virus encoding the T7 RNA polymerase (vTF7.5); comparable viruses enabling the overexpression of the E9 polymerase and the A20 protein have previously been generated in our laboratory (5,14). The availability of these viruses enabled the overexpression of fUDG alone or in combination with A20 and/or the DNA polymerase.…”

“…In this manner, it was determined that processive synthesis requires both the DNA polymerase (E9) and other early viral protein(s) (20), one of which was purified and identified as the product of the A20R gene (5). It is reasonable to hypothesize that UDG might be a second component of the processivity factor.…”