Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

Loregian, Arianna; Sinigalia, Elisa; Mercorelli, Beatrice; Palù, Giorgio; Coen, Donald M.

doi:10.1093/nar/gkm506

Cited by 46 publications

(53 citation statements)

References 48 publications

(93 reference statements)

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“…We also demonstrated that pA104R has a binding site size of around 14 to 16 nt and a minimal binding length of 11 to 20 nt, similar to those reported for other viral DNA-binding proteins (22,23). The results obtained show that pA104R DNAbinding activity is stable at a wide range of temperatures (4°C to 37°C) and pH values (4 to 11), possibly to support ASFV replication in the soft tick vector (Ornithodoros spp.)…”

Section: Discussionsupporting

confidence: 86%

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

Frouco

Freitas

Coelho

et al. 2017

J Virol

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African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Tritoninsoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (Ϫ78.3%), and reduced transcription of a late viral gene (Ϫ47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV.IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

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Section: Discussionsupporting

confidence: 86%

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

Frouco

Freitas

Coelho

et al. 2017

J Virol

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“…6, lanes 3, 7, 9, and 11, respectively). It should be noted that all of the mutants with decreased binding affinity for DNA are not defective in the ability to increase the polymerase processivity, in contrast to the observations for UL42 (39,40) and UL44 (41). In particular, the C95E mutant, which is defective in dimer formation as well as in DNA binding activity, efficiently increased the polymerase processivity, strongly suggesting that the monomeric form of the BMRF1 protein interacts with the BALF5 polymerase catalytic subunit to function as a polymerase processivity factor.…”

Section: Mutations Of the Bmrf1 Protein Affect Its Ability To Increascontrasting

confidence: 59%

“…Although there is no complex structure between DNA and a virus polymerase processivity factor available thus far, site-directed mutation analyses of HSV-1 UL42 supported the proposal that protein-DNA interactions occur with the positively charged amino acid residues on the "back" face (40). The mutational analyses in EBV BMRF1 also demonstrated that substitutions of positively charged residues reduce the DNA-binding affinity (see under "Results").…”

Section: Discussionmentioning

confidence: 88%

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

Murayama

Nakayama

Kato-Murayama

et al. 2009

Journal of Biological Chemistry

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The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-⌬C) was solved in an oligomeric state. The molecular structure of BMRF1-⌬C shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-⌬C, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-⌬C exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-⌬C associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-⌬C probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-⌬C is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.The Epstein-Barr virus (EBV), 4 a human herpesvirus harboring a 172-kbp dsDNA genome, is associated with several B-cell and epithelial cell malignancies and can choose between two alternative life cycles, latent and lytic infection (1). The EBV genomes are replicated as circular plasmid molecules, using the cellular replication machinery of the host in the latent phase of the viral life cycle. On the other hand, after the induction of lytic viral replication, the EBV genome is amplified 100 -1,000-fold by the viral replication machinery. The replication intermediates are large head-to-tail concatemers resulting from rolling-circle DNA replication initiated from oriLyt (2). The EBV replication machinery consists of seven viral gene products (3) as follows: the BZLF1 protein, an oriLytbinding protein; the BALF5 protein, a DNA polymerase catalytic subunit; the BMRF1 protein, a polymerase processivity factor; the BALF2 protein, a single-stranded DNA-binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, putative helicase, primase, and helicase-primase-associated proteins, respectively. It has been suggested that all of the proteins, except for the BZLF1 protein, work together at replication forks to synthesize the leading and lagging strands of the concatemeric EBV genome (2). The EBV DNA polymerase holoenzy...

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“…It has been demonstrated elsewhere that UL84 is capable of binding DNA and RNA (6) and that UL44 binds to DNA (20,22,39). DNA-binding proteins can associate during IP due to their adjacent binding on DNA rather than due to proteinprotein interactions (18,38).…”

Section: Identification Of Proteins Immunoprecipitating With Ul44 Fromentioning

confidence: 99%

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

Strang

Sinigalia

Silva

et al. 2009

J Virol

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The central enzyme responsible for human cytomegalovirus (HCMV) DNA synthesis is a virally encoded DNA polymerase that includes a catalytic subunit, UL54, and a homodimeric accessory subunit, UL44, the presumptive HCMV DNA polymerase processivity factor. The structure of UL44 is similar to that of the eukaryotic processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous other proteins required for faithful DNA replication. We sought to determine whether, like PCNA, UL44 is capable of interacting with multiple DNA replication proteins and, if so, whether these proteins bind UL44 at the site corresponding to where multiple proteins bind to PCNA. Initially, several proteins, including the viral DNA replication factors UL84 and UL57, were identified by mass spectrometry in immunoprecipitates of UL44 from infected cell lysate. The association of UL44/UL84, but not UL44/UL57, was confirmed by reciprocal coimmunoprecipitation of these proteins from infected cell lysates and was resistant to nuclease treatment. Yeast two-hybrid analyses demonstrated that the substitution of residues in UL44 that prevent UL44 homodimerization or abrogate the binding of UL54 to UL44 do not abrogate the UL44/UL84 interaction. Reciprocal glutathione-S-transferase (GST) pulldown experiments using bacterially expressed UL44 and UL84 confirmed these results and, further, demonstrated that a UL54-derived peptide that competes with UL54 for UL44 binding does not prevent the association of UL84 with UL44. Taken together, our results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site. Thus, UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.Protein-protein interactions orchestrate the process of DNA synthesis. Most replicative DNA polymerases include at least two interacting components, a catalytic subunit responsible for the polymerization of DNA and a processivity subunit. The processivity subunit of the polymerase holds the catalytic subunit on DNA while DNA polymerization takes place, thereby permitting long-chain DNA synthesis. One of the best-described processivity factors is proliferating cell nuclear antigen (PCNA) of eukaryotic DNA polymerases ␦ and ε (23, 26). PCNA is a head-to-tail homotrimer which, with the aid of clamp loader proteins, forms a toroidal ring around DNA, creating an internal channel of sufficient diameter to accommodate the DNA duplex (15). Studies of PCNA by numerous laboratories have described a large number of interactions between PCNA and proteins that participate in and abet DNA synthesis (reviewed in references 23 and 26). Of particular interest is that many of these proteins bind PCNA at a specific site, inserting a conserved hydrophobic domain (a PCNA-interacting protein [PIP] box) into a hydrophobic pocket lying beneath an interdomain connector loop of PCNA. PIP box proteins bind and dissociate from PCNA at different times during DNA replication when the need for their funct...

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Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

Cited by 46 publications

References 48 publications

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

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