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. 2008 Dec;82(24):12574-9.
doi: 10.1128/JVI.01193-08. Epub 2008 Oct 8.

Role of homodimerization of human cytomegalovirus DNA polymerase accessory protein UL44 in origin-dependent DNA replication in cells

Elisa Sinigalia  1 , Gualtiero Alvisi, Beatrice Mercorelli, Donald M Coen, Gregory S Pari, David A Jans, Alessandro Ripalti, Giorgio Palù, Arianna Loregian
Affiliations

Role of homodimerization of human cytomegalovirus DNA polymerase accessory protein UL44 in origin-dependent DNA replication in cells

Elisa Sinigalia et al. J Virol. 2008 Dec.

Abstract

The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy.

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Figures

FIG. 1.
FIG. 1.
The UL44-F121A and -L86A/L87 mutants are impaired in their ability to relocalize GFP-UL44ΔNLS to the cell nucleus but can still bind UL54 in living cells. (A) COS-7 cells were transfected to express GFP-UL44ΔNLS alone or in the presence of the indicated DsRed2-UL44 mutant derivatives and imaged by CLSM 24 to 30 h after transfection. Merged images of the green (GFP) and red (DsRed2) channels are shown on the right, with yellow coloration indicative of colocalization. (B) COS-7 cells were transfected to express the indicated DsRed2-UL44 mutant derivatives in the presence of GFP-UL54(1213-1242) and imaged by CLSM 24 to 30 h after transfection. Merged images of the green (GFP) and red (DsRed2) channels are shown on the right, with yellow coloration indicative of colocalization. (C) Results for the determination of the Fn/c values for GFP-UL44ΔNLS in the absence (white bar) or the presence (black bars) of the indicated DsRed2-UL44 fusion proteins, where confocal images such as those shown in panel A were analyzed using Image J 1.62 software. Data represent the means ± standard errors of the means (n > 30). A significant difference (P < 0.05) between the Fn/c values relative to GFP-UL44ΔNLS expressed in the presence of the indicated DsRed2-UL44 mutant derivatives and to GFP-UL44ΔNLS expressed in the presence of wild-type DsRed2-UL44 is indicated by an asterisk. NS, not significant.
FIG. 2.
FIG. 2.
Altered intranuclear localization of UL44 dimerization-defective mutants. (A) COS-7 cells were transfected to express the indicated GFP-UL44 fusion proteins and imaged by CLSM 24 to 30 h after transfection using a 60× water immersion objective. (B) Percentage of cells in which the expression of the indicated GFP-UL44 fusion protein resulted in the formation of nuclear speckles. Data represent the means ± standard errors of the means of the results from three independent experiments (n > 100). A significant difference (P < 0.05) between the values relative to the indicated fusion proteins and that relative to GFP-UL44 is indicated by an asterisk. (C) Quantitative results for the Fn/c ratios of the GFP-UL44 fusion proteins. Confocal images such as those shown in panel A were analyzed as described in the legend to Fig. 1. Data represent the means ± the standard errors of the means (n > 20). A significant difference (P < 0.05) between the Fn/c values relative to the indicated mutant GFP-UL44 fusions and GFP-UL44 is indicated by an asterisk.
FIG. 3.
FIG. 3.
UL44 homodimerization is required for the complementation of HCMV origin-dependent DNA replication. (A) Transient cotransfection-replication assays were performed by transfecting human fibroblasts with the pSP50 plasmid (which contains the HCMV oriLyt DNA replication origin), a plasmid expressing wild-type or mutant full-length UL44 (as indicated on the top of the panel), and a set of plasmids expressing all other essential HCMV replication proteins. Shown is a representative example of the resulting Southern blot analysis of replicated DNA. The position of DpnI-resistant replication products is indicated by an arrow. (B) Lysates of human fibroblasts transfected to express the indicated UL44 proteins or not transfected (mock) were harvested 48 h posttransfection, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Wild-type and mutant UL44 proteins were detected by Western blot analysis with a monoclonal antibody against UL44. Purified baculovirus-expressed UL44 (bv UL44) is also shown as a control.
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