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...

The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ϳ1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.The faithful and efficient duplication of genomic DNA is one of the most conserved processes across all forms of life. Although this is a necessary and highly regulated process, it seems that each model organism has evolved unique modifications during this process. Members of the poxvirus family, of which variola virus is the most notable member and vaccinia virus is the experimental prototype, are no exception. Poxviruses are unique in that they complete the replication and maturation of their ϳ200-kb double-stranded DNA genome in the cytoplasm of the infected host cell. This autonomy dictates that poxviruses encode many of the proteins necessary for nucleotide precursor synthesis and metabolism as well as the core set of enzymes and DNA binding proteins that act directly at the replication fork (1, 2). Indeed, genetic, genomic, and biochemical analysis has revealed that eight proteins are responsible for vaccinia virus DNA synthesis and maturation. This repertoire includes the catalytic DNA polymerase (E9 (3-11)), a stoichiometric component of the heterodimeric processivity factor (A20 (12-15)), a second component of the processivity factor (D4) that also possesses uracil DNA glycosylase (UDG) 2 activity (16 -18), a putative superfamily III helicase with known NTPase and DNA primase activity (D5 (19 -23)), a serine/threonine protein kinase (B1 (24 -26)), an abundant phosphoprotein with essential roles in viral replication, transcription, and morphogenesis (H5 ...

The previously unstudied vaccinia virus gene I2L is conserved in all orthopoxviruses. We show here that the 8-kDa I2 protein is expressed at late times of infection, is tightly associated with membranes, and is encapsidated in mature virions. We have generated a recombinant virus in which I2 expression is dependent upon the inclusion of tetracycline in the culture medium. In the absence of I2, the biochemical events of the viral life cycle progress normally, and virion morphogenesis culminates in the production of mature virions. However, these virions show an ϳ400-fold reduction in specific infectivity due to an inability to enter target cells. Several proteins that have been previously identified as components of an essential entry/fusion complex are present at reduced levels in I2-deficient virions, although other membrane proteins, core proteins, and DNA are encapsidated at normal levels. A preliminary structure/ function analysis of I2 has been performed using a transient complementation assay: the C-terminal hydrophobic domain is essential for protein stability, and several regions within the N-terminal hydrophilic domain are essential for biological competency. I2 is thus yet another component of the poxvirus virion that is essential for the complex process of entry into target cells.Variola virus and vaccinia virus are perhaps the most notorious members of the poxvirus family: the former is the etiological agent of smallpox, and the latter has long been used as the vaccine to protect against smallpox. Poxviruses are the only DNA viruses whose life cycle is restricted to the cytoplasm of infected cells; the large repertoire of proteins encoded by the ϳ200-kb DNA genome of these viruses affords them a high degree of genetic and physical autonomy from host cells. The virions themselves are ϳ250 by 350 nm in size and contain ϳ75 proteins which localize to a delimiting and protein-rich membrane, two proteinaceous lateral bodies of unknown function, and a central core (9). The core, in addition to its numerous structural components, contains the viral genome and a transcriptional apparatus that is responsible for mediating early gene expression immediately after virion binding and entry. The binding of virions to target cells involves interactions between several virion membrane proteins (A27, D8, and H3) and glycosaminoglycans (GAGs) on the target cell (4,5,7,(21)(22)(23)29) and is also presumed to involve higher-affinity interactions between as-yet-unidentified virion proteins and cellular receptors. Subsequent release of the virion core into the cytoplasm can occur either by direct fusion of the virion and plasma membranes or by engulfment of the intact virion followed by pH-dependent fusion events which result in the release of the core from the endocytic compartment and its deposition in the cytoplasm (4,31,51,54).Approximately 90 genes are conserved within the genomes of all chordopoxviruses and are therefore viewed as encoding the repertoire of proteins essential for completion of the viral life cycle (...

In Saccharomyces cerevisiae, ASH1 mRNA is localized to the tip of daughter cells during anaphase of the cell cycle. ASH1 mRNA localization is dependent on four cis-acting localization elements as well as Myo4p, She2p, and She3p. Myo4p, She2p, and She3p are hypothesized to form a heterotrimeric protein complex that directly transports ASH1 mRNA to daughter cells. She2p is an RNA-binding protein that directly interacts with ASH1 cis-acting localization elements and associates with She3p. Here we report the identification of seven She2p mutants-N36S, R43A, R44A, R52A, R52K, R63A, and R63K-that result in the delocalization of ASH1 mRNA. These mutants are defective for RNA-binding activity but retain the ability to interact with She3p, indicating that a functional She2p RNA-binding domain is not a prerequisite for association with She3p. Furthermore, the nuclear/cytoplasmic distribution for the N36S and R63K She2p mutants is not altered, indicating that nuclear/cytoplasmic trafficking of She2p is independent of RNA-binding activity. Using the N36S and R63K She2p mutants, we observed that in the absence of She2p RNA-binding activity, neither Myo4p nor She3p is asymmetrically sorted to daughter cells. However, in the absence of She2p, Myo4p and She3p can be asymmetrically segregated to daughter cells by artificially tethering mRNA to She3p, implying that the transport and/or anchoring of the Myo4p/She3p complex is dependent on the presence of associated mRNA.

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus.