Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Sep;78(17):9203-14.
doi: 10.1128/JVI.78.17.9203-9214.2004.

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication

Kelly S Colletti  1 , Yiyang Xu, Sylvia A Cei, Margaret Tarrant, Gregory S Pari
Affiliations

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication

Kelly S Colletti et al. J Virol. 2004 Sep.

Abstract

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Purification of recombinant UL84 from mammalian cells. Vero cells were infected with a recombinant adenovirus expressing FLAG-tagged UL84. Total protein cell lysates were loaded onto a column containing FLAG affinity beads, and recombinant protein was eluted by using 3X FLAG peptide. Lanes: 1, protein lysate from Ad84-infected Vero cells; 2, column flowthrough; 3, wash; 4, first protein elution fraction with 3X FLAG peptide; 5, second protein elution fraction with 3X FLAG peptide; 6, third protein elution fraction with 3X FLAG peptide; 7, fourth protein elution fraction with 3X FLAG peptide; 8, fifth protein elution fraction with 3X FLAG peptide.
FIG. 2.
FIG. 2.
Generation of a monoclonal antibody to UL84 (MAb84). Purified UL84 recombinant protein isolated from Cos7 cells was used to produce a monoclonal antibody. (A) Western blot of cell protein lysates from either the transfection of various UL84 expression constructs or HCMV infection and reacted with MAb84. Lanes: 1-586(FL), cell lysate from Cos7 cells transfected with pUL84-TAG(1-586); 84-EGFP, cell lysate from Cos7 cells transfected with a plasmid expressing a UL84-EGFP fusion protein; TAG-Control, cell lysate from Cos7 cells transfected with a pFLAG-control expression plasmid; AD169, cell lysate from human foreskin fibroblasts infected with HCMV (AD169) harvested 72 h postinfection. (B) UL84 deletion constructs used to map the epitope for MAb84. (C) Western blot of MAb84 reacted with a series of UL84 deletion mutants. Lanes: 1, cell lysate from Cos7 cells transfected with UL84 deletion mutant pUL84-TAG(83-586); 2, cell lysate from Cos7 cells transfected with UL84 deletion mutant pUL84-TAG(104-586); 3, cell lysate from Cos7 cells transfected with UL84 deletion mutant pUL84-TAG(141-586); 4, cell lysate from Cos7 cells transfected with UL84 deletion mutant pUL84-TAG(201-586); 5, cell lysate from Cos7 cells transfected with UL84 deletion mutant pUL84-TAG(351-586); 6, cell lysate from Cos7 cells transfected with UL84 deletion mutant pUL84-TAG(1-513). (D) MAb84 interacts with a region of the UL84 protein mapping to aa 101 to 200. Cos7 cells were transfected with expression plasmids expressing ∼100-aa segments of the UL84 protein. Each segment was constructed such that it was fused in frame with the FLAG epitope. Cell lysates were prepared and a Western blot was performed by using MAb84. Lanes correspond to each expression plasmid. Panel anti-UL84 was reacted with MAb84 antibody. Panel anti-FLAG was reacted with anti-FLAG antibody.
FIG. 3.
FIG. 3.
UL84 interacts with itself in vivo. (A) Western blot of coimmunoprecipitated cell lysates transfected with pUL84-EGFP and pUL84-TAG(1-586). Lanes: 1, pUL84-TAG(1-586) and pUL84-EGFP cotransfected cell lysates immunoprecipitated with anti-FLAG and reacted with anti-EGFP; 2, pUL84-FLAG and pEGFP-N1 cotransfected cell lysates immunoprecipitated with anti-FLAG and reacted with anti-EGFP; 3, pUL84-TAG(1-586) and pUL84-EGFP cotransfected cell lysates immunoprecipitated with anti-EGFP and reacted with anti-FLAG; 4, FLAG-control plasmid and pUL84-EGFP cotransfected cell lysates immunoprecipitated with anti-EGFP and reacted with anti-FLAG. (B) Western blot of coimmunoprecipitates from cell lysates containing UL84-FLAG deletion proteins and full-length UL84-HA-tagged protein. Lanes: FL, mixed lysates from cells transfected with pUL84-TAG(1-586) and pUL84-HA; 1, mixed lysates from cells transfected with pUL84-TAG(83-586) and pUL84-HA; 2, mixed lysates from cells transfected with pUL84-TAG(104-586) and pUL84-HA; 3, mixed lysates from cells transfected with pUL84-TAG(141-586) and pUL84-HA; 4, mixed lysates from cells transfected with pUL84-TAG(201-586) and pUL84-HA. The top panel shows a Western blot of coimmunoprecipitated protein reacted with anti-FLAG antibody. The bottom panel shows a Western blot of coimmunoprecipitated protein reacted with anti-HA antibody.
FIG. 4.
FIG. 4.
The UL84 oligomerization domain maps to aa 151 to 200 and does not involve the leucine zipper motif. (A) Schematic of UL84 ORF and FLAG-tagged peptides used to define the protein domain involved in UL84 oligomerization and the relative positions of the leucine zipper motif (LZ) and charged region (CR). Fragments labeled 1, 2, 3, and 4 correspond to constructs p84FR(1-100), p84FR(101-200), p84FR(201-300), and p84OD(151-300), respectively. (B) UL84 oligomerization involves a region of the protein mapping between aa 101 and 200. Cell lysates transfected with plasmids expressing small peptides of UL84 were incubated with cell lysates prepared from cells transfected with full-length UL84 (pUL84EXPRESS). The top panel shows a Western blot of cell lysates prepared from cells transfected with pUL84EXPRESS and reacted with MAb84. Lanes in the middle panel show the following: lane 1, mixed cell lysates from cells transfected with pUL84EXPRESS and p84FR(1-100), immunoprecipitated, with anti-FLAG, and reacted with anti-FLAG; lane 2, mixed cell lysates from cells transfected with pUL84EXPRESS and p84FR(101-200), immunoprecipitated with anti-FLAG, and reacted with anti-FLAG; lane 3, mixed cell lysates from cells transfected with pUL84EXPRESS and p84FR(201-300), immunoprecipitated with anti-FLAG, and reacted with anti-FLAG. Lanes in the bottom panel show: lane 1, mixed cell lysates from cell transfected with pUL84EXPRESS and p84FR(1-100), immunoprecipitated with anti-FLAG, and reacted with MAb84; lane 2, mixed cell lysates from cell transfected with pUL84EXPRESS and p84FR(101-200), immunoprecipitated with anti-FLAG, and reacted with MAb84; lane 3, mixed cell lysates from cell transfected with pUL84EXPRESS and p84FR(201-300), immunoprecipitated with anti-FLAG, and reacted with MAb84. (C) The UL84 OD is located between aa 151 and 200. A Western blot of coimmunoprecipitated protein from mixed cell lysates from cells transfected with pUL84-HA and p84OD(151-300) (polypeptide 4) expression plasmids is shown. (Top panel) Western blot reacted with anti-HA antibody. Lanes: 1, lysate from cells transfected with a UL84-HA expression plasmid; 2, immunoprecipitated protein from mixed cell lysates with anti-FLAG antibody and reacted with anti-HA antibody on a Western blot. (Bottom panel) Western blot reacted with anti-FLAG antibody. Lanes: 1, protein lysate from cells transfected with a p84OD(151-300) (polypeptide 4); 2, immunoprecipitated protein from mixed cell lysates with anti-FLAG antibody and reacted with anti-FLAG antibody. (D) UL84 forms a homodimer in vitro. UL84 expression plasmids were used in vitro translation reactions, and the resulting polypeptides were subjected to chemical cross-linking. Lanes: 1, in vitro-translated protein produced from p84OD(151-300); 2, in vitro-translated protein produced from p84OD(151-300) incubated with 0.008% glutaraldehyde; 3, in vitro-translated protein produced from pIE2-HA(387-580); 4, in vitro-translated protein produced from pIE2-HA(387-580) incubated with 0.008% glutaraldehyde.
FIG. 5.
FIG. 5.
The leucine zipper motif of UL84 is not involved in oligomerization but is required for oriLyt-dependent DNA replication. (A) Schematic of the UL84 leucine zipper motif, indicating leucine residues 1 (aa 114), 2 (aa 121), 3 (aa 128), and 4 (aa 135) that comprise the putative leucine zipper. (B) Coimmunoprecipitation of UL84-HA and UL84 leucine zipper mutants. Mutation of the UL84 leucine zipper motif does not interfere with UL84 oligomerization. UL84 FLAG-tagged leucine zipper mutants were cotransfected with full-length UL84-HA-tagged protein, and protein lysates were immunoprecipitated with anti-FLAG antibody. Coimmunoprecipitated protein was analyzed by Western blotting with anti-HA (top panel) and anti-FLAG (bottom panel) antibodies. (Top panel) All lanes contain coimmunoprecipitated UL84-HA protein. (Bottom panel) Lanes: L2+L3, UL84 leucine zipper mutant in which leucine residues 2 and 3 were changed to valine; L1+L2, UL84 leucine zipper mutant in which leucine residues 1 and 2 were changed to valine; L3+L4, UL84 leucine zipper mutant in which leucine residues 3 and 4 were changed to valine. (C) An intact UL84 leucine zipper is required for complementation of oriLyt-dependent DNA replication. A cotransfection replication assay was performed by using UL84 leucine zipper mutants in place of wild-type UL84. T-HFs were cotransfected with the core replication proteins plus IE2 and oriLyt. Total cell DNA was harvested and cleaved with EcoRI and DpnI, and replicated oriLyt DNA was analyzed by Southern blotting. Shown is the replicated oriLyt band. Lanes: Control, DNA from cells cotransfected with the core replication proteins plus IE2, oriLyt, and pUL84-TAG(1-586); L2+L3, same as the control except the UL84 leucine zipper mutant, UL84 L2+L3, was substituted for pUL84-TAG(1-586); L1+L2, same as the control except the UL84 leucine zipper mutant, UL84 L1+L2, was substituted for pUL84-TAG(1-586); L3+L4, same as the control except the UL84 leucine zipper mutant, UL84 L3+L4, was substituted for pUL84-TAG(1-586).
FIG. 6.
FIG. 6.
UL84 leucine zipper mutants no longer repress IE2-mediated transactivation. Graph of luciferase reporter assay showing the effects various UL84 leucine zipper mutants on the IE2-mediated transactivation of the UL112/113 promoter. Vero cells were cotransfected with the luciferase reporter construct p112/113pr, pON2206, and either pUL84-TAG(91-586) or the mutated leucine zipper constructs pUL84 L1+L2, pUL84 L2+L3, or pUL83 L3+L4. Error bars indicate the standard deviations of three separate experiments.
FIG. 7.
FIG. 7.
An intact UL84 leucine zipper motif is required for interaction with IE2. Cos7 cells were cotransfected with the IE2 expression construct pON2206 and UL84 leucine zipper mutant L1+L2. Cell lysates were immunoprecipitated with anti-FLAG antibody and coimmunoprecipitates were analyzed by Western blot with C13-12E2 and anti-FLAG antibodies. Lanes: 1, coimmunoprecipitation of wild-type IE2 (top panel) and UL84-TAG(1-586) protein (bottom panel); 2, immunoprecipitation of wild-type IE2 (top panel) and UL84 L1+L2 protein (bottom panel); 3, control cell lysates containing IE2 (top panel) and UL84 L1+L2 protein (bottom panel) reacted with C13-12E2 and anti-FLAG, respectively.
FIG. 8.
FIG. 8.
UL84 interacts with a protein domain within the first 215 aa of IE2. DNA fragments of IE2 cDNA were subcloned into an expression vector such that the region of the IE2 protein would be expressed in frame with the HA epitope and the SV40 NLS. These constructs were transfected into Cos7 cells and lysates were prepared and mixed with pUL84-TAG(1-586) protein cell lysates. The mixed cell lysates were immunoprecipitated with anti-FLAG. Coimmunoprecipitates were analyzed by Western blot with anti-HA antibody. (A) Schematic of IE2 expression constructs used for coimmunoprecipitations. (B) Coimmunoprecipitation of UL84 and IE2 peptides. (Top panel) Western blot of cell lysates transfected with IE2 expression plasmids with anti-HA. Lanes: 1, protein lysates from cells transfected with pIE2-HA(1-215); 2, protein lysates from cells transfected with plasmid pIE2-HA(216-386); 3, protein lysates from cells transfected with plasmid pIE2-HA(387-580). (Middle panel) Lanes 1 to 3 are protein lysates from cells transfected with pUL84-TAG(1-586) with anti-FLAG. (Bottom panel) Coimmunoprecipitation of IE2 peptide 1-215 and full-length UL84. Western blot analysis of coimmunoprecipitates from mixed cell lysates containing expressed proteins. Mixed lysates were immunoprecipitated with anti-FLAG antibody and reacted with anti-HA antibody. Lanes: 1, coimmunoprecipitation of mixed lysates from cells expressing pIE2-HA(1-215) and pUL84-TAG(1-586); 2, coimmunoprecipitation of mixed lysates from cells expressing pIE2-HA(216-386) and pUL84-TAG(1-586); 3, coimmunoprecipitation of mixed lysates from cells expressing pIE2-HA(387-580) and pUL84-TAG(1-586). (C) Mixed lysate coimmunoprecipitation of UL84 and IE2. Protein lysates containing pON2206 and pUL84-TAG(1-586) were mixed together and immunoprecipitated with anti-FLAG. Coimmunoprecipitated protein analyzed by Western blot by using either G13-12E2 (right panel) or anti-FLAG (left panel).
FIG. 9.
FIG. 9.
Transdominant-negative inhibition of oriLyt-dependent DNA replication. T-HFs were cotransfected with the HHV-8 core replication expression plasmids plus pON2206, pUL84-TAG(1-586), HCMV oriLyt, and various UL84 and IE2 plasmids expressing small peptides. DNA was harvested and cleaved with EcoRI and DpnI. Replication products were examined by Southern blot analysis. (A) Expression of UL84 aa 101 to 200 inhibits oriLyt-dependent DNA replication. Lanes: Control, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt; +p84FR(1-100), cotransfection of core replication proteins, pON2206, pUL84 TAG, and HCMV oriLyt plus construct p84FR(1-100); +p84FR(101-200), cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt plus construct p84FR(101-200); +p84FR(201-300), cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt plus construct p84FR(201-300); +p84FR(301-400), cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586) and HCMV oriLyt plus construct p84FR(301-400); +p84FR(401-500), cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586) and HCMV oriLyt plus construct p84FR(401-500); +p84FR(501-586), cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586) and HCMV oriLyt plus construct p84FR(501-586). (B) Expression of IE2 aa 1 to 215 inhibits oriLyt-dependent DNA replication. Lanes: 1, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt; 2, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt plus construct pIE2-HA(1-215); 3, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt plus construct pIE2-HA(216-386); 4, cotransfection of core replication proteins, pON2206, pUL84-TAG (1-586), and HCMV oriLyt plus construct pIE2-HA(387-580). (C) Expression of a peptide containing an intact oligomerization domain and a mutated leucine zipper motif (p84-2LZA) inhibits oriLyt-dependent DNA replication. Lanes: 1, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt; 2, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt plus construct p84-2LZA; 3, cotransfection of core replication proteins, pON2206, pUL84-TAG(1-586), and HCMV oriLyt plus construct p84OD(151-300).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. AuCoin, D. P., K. S. Colletti, S. A. Cei, I. Papouskova, M. Tarrant, and G. S. Pari. 2004. Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP). Virology 318:542-555. - PubMed
    1. Boehmer, P. E., and I. R. Lehman. 1993. Physical interaction between the herpes simplex virus 1 origin-binding protein and single-stranded DNA-binding protein ICP8. Proc. Natl. Acad. Sci. USA 90:8444-8448. - PMC - PubMed
    1. Brandt, S., P. Thorkildson, and T. R. Kozel. 2003. Monoclonal antibodies reactive with immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans. Clin. Diagn. Lab. Immunol. 10:903-909. - PMC - PubMed
    1. Chang, Y. N., D. L. Dong, G. S. Hayward, and S. D. Hayward. 1990. The Epstein-Barr virus Zta transactivator: a member of the bZIP family with unique DNA-binding specificity and a oligomerization domain that lacks the characteristic heptad leucine zipper motif. J. Virol. 64:3358-3369. - PMC - PubMed
    1. Chen, D., and P. D. Olivo. 1994. Expression of the varicella-zoster virus origin-binding protein and analysis of its site-specific DNA-binding properties. J. Virol. 68:3841-3849. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources