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-encoded D5 protein is an essential ATPase involved in viral DNA replication. We have expanded the genotypic and phenotypic analysis of six temperature-sensitive (ts) D5 mutants (Cts17, Cts24, Ets69, Dts6389 [also referred to as Dts38], Dts12, and Dts56) and shown that at nonpermissive temperature all of the tsD5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For Cts17 and Cts24, this restriction reflects the thermolability of the D5 proteins. The Dts6389, Dts12, and Dts56 D5 proteins become insoluble at 39.7°C, while the Ets69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7°C. To investigate which structural features of D5 are important for its biological and biochemical activities, we generated targeted mutations in invariant residues positioned within conserved domains found within D5. Using a transient complementation assay that assessed the ability of D5 variants to sustain ongoing DNA synthesis during nonpermissive Cts24 infections, only a wtD5 allele supported DNA synthesis. Alleles of D5 containing targeted mutations within the Walker A or B domains, the superfamily III helicase motif C, or the AAA؉ motif lacked biological competency. Furthermore, purified preparations of these variant proteins revealed that they all were defective in ATP hydrolysis. Multimerization of D5 appeared to be a prerequisite for enzymatic activity and required the Walker B domain, the AAA؉ motif, and a region located upstream of the catalytic core. Finally, although multimerization and enzymatic activity are necessary for the biological competence of D5, they are not sufficient.Vaccinia virus is the prototypic member of the poxvirus family, whose most infamous member is the causative agent of smallpox, variola virus. Vaccinia virus encodes ϳ200 gene products and replicates solely within the cytoplasm of the infected cell. Vaccinia virus is thought to encode most, if not all, of the proteins involved in the faithful and robust duplication of its 192-kb DNA genome. Genetic, genomic, and biochemical studies have elucidated five proteins as being essential for vaccinia virus DNA replication. These include the viral DNA polymerase (E9 [2, 3, 25-27, 38, 40, 41, 45]), the heterodimeric processivity factor (A20/D4 [16,32,39]), a serine/threonine protein kinase (B1 [34,35,43]), and a DNA-independent nucleoside triphosphatase (NTPase; D5 [9,10]). D5 is a 90-kDa protein that is made at early times postinfection, with the peak synthesis coinciding with the onset of viral DNA synthesis (10). We have previously described a protocol for in vivo overexpression and subsequent purification of D5 and demonstrated that it possesses nucleic acid-independent NTPase activity (9). From the three original collections of vaccinia virus temperature-sensitive (ts) mutants, the Condit collection (4), the Ensinger collection (8), and the Dales collection (5), six mutants with lesions that map to the D5 open reading frame (ORF) have been des...

Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.

TMEM27 (collectrin), a homologue of angiotensin converting enzyme 2 (ACE2), was previously shown in renal proximal tubule to regulate amino acid uptake via interaction with the broad range neutral amino acid transporter B0AT1, whereas ACE2 was shown to associate with B0AT1 in small intestine. To investigate the role of collectrin interaction with B0AT1, we generated Madin‐Darby canine kidney (MDCK) cell lines constitutively expressing B0AT1 and observed that this expression was not tolerated. Therefore, using a KRAB repressor system and lentiviral transduction, we produced cells that inducibly express either B0AT1, collectrin or both. As in vivo, collectrin was shown to be required for efficient B0AT1 cell surface expression and function. Furthermore, we demonstrated that collectrin co‐expression increases B0AT1 protein stability. Interestingly, expression levels of both B0AT1 and collectrin were increased when amino acid concentrations in the culture medium were reduced to physiological levels. In conclusion, we generated cell lines expressing inducible B0AT1 and collectrin recapitulating the dependence of B0AT1 expression on collectrin co‐expression and thus representing a very good model for testing the role and the mechanism of B0AT1‐collectrin interaction and the impact of the amino acid concentration for the control of its expression.