A Mutation in the C-terminal Putative Zn2+ Finger Motif of UL52 Severely Affects the Biochemical Activities of the HSV-1 Helicase-Primase Subcomplex

Biswas, Nilima; Weller, Sandra K.

doi:10.1074/jbc.274.12.8068

Cited by 43 publications

(90 citation statements)

References 55 publications

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“…While the restriction we observed is likely to occur independently of ICP0 (Fig. 4A and B), we note that other viral and cellular regulators have been reported to bind Zn 2ϩ (2,24,28). It is likely therefore that depletion of intracellular Zn 2ϩ would inhibit multiple regulators required for HSV-1 replication.…”

mentioning

confidence: 66%

Depletion of Intracellular Zinc Inhibits the Ubiquitin Ligase Activity of Viral Regulatory Protein ICP0 and Restricts Herpes Simplex Virus 1 Replication in Cell Culture

Grant

Tong

et al. 2012

J Virol

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The viral ubiquitin ligase ICP0 stimulates the onset of HSV-1 lytic infection and productive reactivation of viral genomes from latency. In order to mediate these processes, it requires its C3HC4 RING finger domain, a tertiary structural fold that is coordinated by the binding of two zinc (Zn 2؉ ) atoms. Here we formally demonstrate that Zn 2؉ binding and intracellular Zn 2؉ levels are critical for ICP0's biochemical activity and that depletion of intracellular Zn 2؉ severely attenuates HSV-1 replication. Herpes simplex virus type-1 (HSV-1) is a prevalent human pathogen whose clinical importance is accentuated by its ability to attain and periodically reactivate from a latent state of infection (reviewed in reference 7). A key regulator in this process is ICP0, a viral RING finger ubiquitin ligase (4,10,15,26) that is required for the efficient initiation of lytic replication and productive reactivation of viral genomes from latency (reviewed in reference 26). While studies have shown that mutations of Zn 2ϩ coordinating residues within the RING domain of ICP0 impair its ability to stimulate infection and viral reactivation (18,19,22), a recent study demonstrated that a viral mutant expressing ICP0-RF, a double Zn 2ϩ coordinating RING finger mutant (Fig. 1A), replicated with greater efficiency than an ICP0 null mutant virus (18). This observation implies that ICP0 Zn 2ϩ binding RING mutants may retain a minimal degree of functional activity. ICP0-RF was also reported to reduce viral reactivation efficiency when coexpressed with an ICP0 C-terminal truncation mutant in trans following superinfection (18), suggesting that specific ICP0 RING mutants may also have transdominant or corepressor properties. The majority of studies correlating the contribution of ICP0's biochemical ubiquitin ligase activity to that of its functional requirement during infection have been conducted using the RING deletion mutant ICP0-FXE (4, 8-10), a mutant which lacks the majority of the secondary structural features of the RING domain (Fig. 1A). Viral mutants that express ICP0-FXE have replication and reactivation defects equivalent to those of an ICP0 null mutant (8,9,16,20). We therefore decided to compare the biochemical and biological properties of ICP0-FXE, a commonly utilized RING mutant, to those of ICP0-RF in order to determine if they had any significant phenotypic differences.To verify that mutation of Cys116/156 within ICP0-RF (Fig. 1A) was sufficient to inhibit Zn 2ϩ binding and RINGdependent ubiquitin ligase activity, cDNAs encoding the RING domains of ICP0-FXE (8) and -RF (22) were transferred into the bacterial expression vector pGEX241 (4). Wild-type (wt) and mutant RING domains were purified (as described in reference 4) and analyzed for their ability to bind Zn 2ϩ and stimulate polyubiquitin chain formation in vitro. Only the wt RING domain was able to release Zn 2ϩ (Fig. 1B) and catalyze the formation of unanchored polyubiquitin chains (Fig. 1C). To confirm that ICP0-RF was biochemically inactive during infection, HF...

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confidence: 66%

Depletion of Intracellular Zinc Inhibits the Ubiquitin Ligase Activity of Viral Regulatory Protein ICP0 and Restricts Herpes Simplex Virus 1 Replication in Cell Culture

Grant

Tong

et al. 2012

J Virol

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“…The six helicase motifs have been identified, and functional roles have been established by site-directed mutagenesis (51). The UL52 primase has a catalytic DXD motif and an evolutionarily conserved C-terminal zinc finger, and both are required for function (52)(53)(54). The UL8 protein has no apparent cellular homolog that could be identified by sequence analysis.…”

Section: Properties Of Replisome Proteinsmentioning

confidence: 99%

Replication and Recombination of Herpes Simplex Virus DNA

Muylaert

Tang

Elias

2011

Journal of Biological Chemistry

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Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the singlestranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.Herpesviruses are found in all animals from molluscs to man. During evolution, the viruses have become tightly associated and co-evolved with their hosts. They seem to cross species borders only by accident, and in such rare instances, they may cause unexpected and severe disease. Herpesviruses have an unusual lifestyle; they cause lytic infection in cells, leading to efficient production of new infectious virus particles, and they also establish latent infections in either non-dividing neuronal cells or cycling cells of the immune system. The latent state is characterized by expression of a very limited set of genes to ascertain maintenance of virus chromosomes and to escape recognition by the immune system. The mechanisms for establishing latency appear to differ considerably for different herpesviruses. In contrast, mechanisms for replication of virus DNA during lytic infection and subsequent formation of infectious particles seem to be evolutionarily conserved. There is one notable exception; the mechanism for recognition of origins of DNA replication and initiation of DNA synthesis differs between the herpesvirus families.Humans can be infected by eight different herpesviruses. Herpes simplex viruses I and II and varicella zoster virus are alphaherpesviruses. Cytomegalovirus and the roseoloviruses, human herpesviruses 6 and 7, are classified as betaherpesviruses. Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus belong to the gammaherpesvirus subfamily.In this minireview, we discuss recent developments in replication, recombination, and repair of herpes simplex virus DNA. We will take as our starting point a previous minireview in the Journal of Biological Chemistry that provides an insightful and accurate description of basic mechanisms and components of the herpes simplex virus replication machinery (1). Noteworthy new developments have been (i) the presentation of three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein (ssDNA) 2 ; (ii) the recons...

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Section: The Minichromosome Maintenance (Mcm)mentioning

confidence: 99%

“…1) in a nonconserved region of the MCM family of proteins suggests the importance of the zinc finger domain in MCM function (2). Furthermore, mutational analyses of zinc finger domains in several viral helicases have shown these motifs to be important for helicase activity (28,29).Archaea, the third domain of life (30), are believed to replicate DNA in a eukaryotic-like fashion (31, 32). This conclusion is based in large part on the complete genome sequences of several members of this domain and the biochemical properties of the replication proteins that have been studied.…”

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confidence: 99%

The Zinc Finger Domain of the Archaeal Minichromosome Maintenance Protein Is Required for Helicase Activity

Poplawski¹,

Grabowski²,

Long³

et al. 2001

Journal of Biological Chemistry

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The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum ⌬H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX 2 CX n CX 2 C (C 4 ) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif. The minichromosome maintenance (MCM)1 genes encode a family of six proteins (MCM2 to -7) first identified by their essential role in the maintenance of ARS-containing minichromosomes in Saccharomyces cerevisiae (1, 2). MCM homologues have been identified in all eukaryotes, and all are essential for DNA replication (1, 3-7). The recruitment of these proteins onto replication origins during the G 1 phase of the cell cycle is essential for the formation of a prereplicative complex and initiation of DNA synthesis (8 -12). In addition to the heterohexameric structure MCM2/3/4/5/6/7, found in vivo and in vitro, several additional MCM complexes have been identified (13-16). Biochemical studies in yeast and mammals have shown that a dimeric complex of the MCM4/6/7 heterotrimer contains 3Ј-5Ј DNA helicase activity, single-stranded DNA (ssDNA) binding, and DNA-dependent ATPase activities (15,17,18). Both MCM2 and MCM3/5 were shown to inhibit the helicase activity of MCM4/6/7 and thus were suggested to play regulatory roles (15,19). Both genetic and biochemical data suggest that the MCM4/6/7 complex is the eukaryotic replicative helicase and that the other MCM polypeptides may play regulatory roles (15,16,19).Zinc fingers were first identified as zinc-binding domains important for protein-DNA interactions (20, 21). They contain conserved Cys and His residues, and to date more than 10 classes have been biochemically characterized, and their structures have been determined (reviewed in Refs. 22-25). Since their discovery, zinc finger domains have been implicated in diverse functions, including ssDNA and double-stranded DNA (dsDNA) binding, RNA binding, and protein-protein interactions (reviewed in Refs. 24 and 26). The primary amino acid sequences of the eukaryotic MCM2, -4, -6, and -7 proteins from all organisms contain a putative zinc finger motif of the C 4 type, either CX 2 CX n CX 2 C (MCM2) or CX 2 CX n CX 4 C (MCM4, -6, an...

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A Mutation in the C-terminal Putative Zn2+ Finger Motif of UL52 Severely Affects the Biochemical Activities of the HSV-1 Helicase-Primase Subcomplex

Cited by 43 publications

References 55 publications

Depletion of Intracellular Zinc Inhibits the Ubiquitin Ligase Activity of Viral Regulatory Protein ICP0 and Restricts Herpes Simplex Virus 1 Replication in Cell Culture

Depletion of Intracellular Zinc Inhibits the Ubiquitin Ligase Activity of Viral Regulatory Protein ICP0 and Restricts Herpes Simplex Virus 1 Replication in Cell Culture

Replication and Recombination of Herpes Simplex Virus DNA

The Zinc Finger Domain of the Archaeal Minichromosome Maintenance Protein Is Required for Helicase Activity

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