Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

Download Full-text

Internal Deletions of IE2 86 and Loss of the Late IE2 60 and IE2 40 Proteins Encoded by Human Cytomegalovirus Affect the Levels of UL84 Protein but Not the Amount of UL84 mRNA or the Loading and Distribution of the mRNA on Polysomes

Journal of Virology ◽

10.1128/jvi.01293-08 ◽

2008 ◽

Vol 82 (22) ◽

pp. 11383-11397 ◽

Cited By ~ 16

Author(s):

Rebecca L. Sanders ◽

Christia J. Del Rosario ◽

Elizabeth A. White ◽

Deborah H. Spector

Keyword(s):

Human Cytomegalovirus ◽

Viral Dna ◽

Viral Dna Replication ◽

Cytoplasmic Transport ◽

Alternatively Spliced ◽

Infected Cells ◽

Efficient Expression ◽

The Stability

ABSTRACT The major immediate-early (IE) region of human cytomegalovirus encodes two IE proteins, IE1 72 and IE2 86, that are translated from alternatively spliced transcripts that differ in their 3′ ends. Two other proteins that correspond to the C-terminal region of IE2 86, IE2 60 and IE2 40, are expressed at late times. In this study, we used IE2 mutant viruses to examine the mechanism by which IE2 86, IE2 60, and IE2 40 affect the expression of a viral DNA replication factor, UL84. Deletion of amino acids (aa) 136 to 290 of IE2 86 results in a significant decrease in UL84 protein during the infection. This loss of UL84 is both proteasome and calpain independent, and the stability of the protein in the context of infection with the mutant remains unaffected. The RNA for UL84 is expressed to normal levels in the mutant virus-infected cells, as are the RNAs for two other proteins encoded by this region, UL85 and UL86. Moreover, nuclear-to-cytoplasmic transport and the distribution of the UL84 mRNA on polysomes are unaffected. A region between aa 290 and 369 of IE2 86 contributes to the UL84-IE2 86 interaction in vivo and in vitro. IE2 86, IE2 60, and IE2 40 are each able to interact with UL84 in the mutant-infected cells, suggesting that these interactions may be important for the roles of UL84 and the IE2 proteins. Thus, these data have defined the contribution of IE2 86, IE2 60, and IE2 40 to the efficient expression of UL84 throughout the infection.

Download Full-text

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.12.2680 ◽

1997 ◽

Vol 41 (12) ◽

pp. 2680-2685 ◽

Cited By ~ 33

Author(s):

D J Tenney ◽

G Yamanaka ◽

S M Voss ◽

C W Cianci ◽

A V Tuomari ◽

...

Keyword(s):

Protein Kinase ◽

Dna Synthesis ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Potent Inhibitor ◽

Viral Dna ◽

Infected Cells ◽

Simplex Virus ◽

Hsv Thymidine Kinase

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

Download Full-text

Nucleolin Associates with the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 and Is Necessary for Efficient Viral Replication

Journal of Virology ◽

10.1128/jvi.01510-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1771-1784 ◽

Cited By ~ 52

Author(s):

Blair L. Strang ◽

Steeve Boulant ◽

Donald M. Coen

Keyword(s):

Infected Cell ◽

Dna Synthesis ◽

Dna Polymerase ◽

Western Blotting ◽

Human Cytomegalovirus ◽

Major Protein ◽

Cell Nuclei ◽

Viral Dna ◽

Infected Cells ◽

Processivity Factor

ABSTRACT In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.

Download Full-text

Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.00189-10 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8409-8421 ◽

Cited By ~ 23

Author(s):

Young-Eui Kim ◽

Jin-Hyun Ahn

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Specific Interaction ◽

Terminal Region ◽

Replication Proteins ◽

Viral Dna ◽

Viral Dna Replication ◽

Carboxy Terminal Region ◽

Processivity Factor ◽

Carboxy Terminal

ABSTRACT The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.

Download Full-text

DNA Polymerase Processivity Factor of Human Cytomegalovirus May Be a Key Molecule for Molecular Coupling of Viral DNA Replication to Transcription

DNA Replication and Related Cellular Processes ◽

10.5772/18144 ◽

2011 ◽

Author(s):

Hiroki Isomura

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Viral Dna ◽

Viral Dna Replication ◽

Processivity Factor

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00663-09 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7581-7589 ◽

Cited By ~ 26

Author(s):

Blair L. Strang ◽

Elisa Sinigalia ◽

Laurie A. Silva ◽

Donald M. Coen ◽

Arianna Loregian

Keyword(s):

Dna Replication ◽

Infected Cell ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Nuclear Antigen ◽

Replication Proteins ◽

Viral Dna ◽

Viral Dna Replication ◽

Processivity Factor ◽

Replication Factors

ABSTRACT 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.

Download Full-text

Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase

Journal of Virology ◽

10.1128/jvi.01875-09 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7459-7472 ◽

Cited By ~ 6

Author(s):

Yali Zhu ◽

Zetang Wu ◽

M. Cristina Cardoso ◽

Deborah S. Parris

Keyword(s):

Dna Polymerase ◽

Wild Type ◽

Viral Dna ◽

Viral Dna Replication ◽

Infected Cells ◽

Simplex Virus ◽

Lagging Strand ◽

Hsv 1

ABSTRACT The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo−) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo− HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo− HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.

Download Full-text

Effect of incorporation of cidofovir into DNA by human cytomegalovirus DNA polymerase on DNA elongation.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.3.594 ◽

1997 ◽

Vol 41 (3) ◽

pp. 594-599 ◽

Cited By ~ 121

Author(s):

X Xiong ◽

J L Smith ◽

M S Chen

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Chain Elongation ◽

Exonuclease Activity ◽

Dna Chain ◽

Alternate Substrate

Cidofovir (CDV) (HPMPC) has potent in vitro and in vivo activity against human cytomegalovirus (HCMV), CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is an inhibitor and an alternate substrate of HCMV DNA polymerase. CDV is incorporated with the correct complementation to dGMP in the template, and the incorporated CDV at the primer end is not excised by the 3'-to-5' exonuclease activity of HCMV DNA polymerase. The incorporation of a CDV molecule causes a decrease in the rate of DNA elongation for the addition of the second natural nucleotide from the singly incorporated CDV molecule. The reduction in the rate of DNA (36-mer) synthesis from an 18-mer by one incorporated CDV is 31% that of the control. However, the fidelity of HCMV DNA polymerase is maintained for the addition of the nucleotides following a single incorporated CDV molecule. The rate of DNA synthesis by HCMV DNA polymerase is drastically decreased after the incorporation of two consecutive CDV molecules; the incorporation of a third consecutive CDV molecule is not detectable. Incorporation of two CDV molecules separated by either one or two deoxynucleoside monophosphates (dAMP, dGMP, or dTMP) also drastically decreases the rate of DNA chain elongation by HCMV DNA polymerase. The rate of DNA synthesis decreases by 90% when a template which contains one internally incorporated CDV molecule is used. The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of HCMV DNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.

Download Full-text

Characterization of Human Herpesvirus 8 ORF59 Protein (PF-8) and Mapping of the Processivity and Viral DNA Polymerase-Interacting Domains

Journal of Virology ◽

10.1128/jvi.74.23.10920-10929.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 10920-10929 ◽

Cited By ~ 38

Author(s):

Szeman Ruby Chan ◽

Bala Chandran

Keyword(s):

Dna Polymerase ◽

Human Herpesvirus ◽

Human Herpesvirus 8 ◽

Dependent Manner ◽

Cell Nuclei ◽

Viral Dna ◽

Herpesvirus 8 ◽

Barr Virus

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118–126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

Download Full-text