Conserved residues in the UL24 protein of herpes simplex virus 1 are important for dispersal of the nucleolar protein nucleolin

Abstract

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39 degrees C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.

FIG. 1.

Substitutions introduced into HSV-1 UL24. (A) The large rectangle represents the UL24 protein. Black boxes illustrate the positions of the five homology domains, numbered one to five in Roman numerals. The different single and double substitutions studied are indicated above or below the rectangle. (B) Expression of the different versions of HA-UL24 as detected by Western blot analysis of lysates prepared from transiently transfected COS-7 cells, using an antibody directed against the HA tag. Positions of molecular mass markers are indicated to the left of the panel.

FIG. 2.

Impact of UL24 variants on the localization of nucleolin. Confocal images of COS-7 cells transiently expressing different forms of HA-UL24 with the indicated amino acid substitutions or with the wild-type UL24 sequence and immunostained for HA-UL24 (green) and nucleolin (red). In each set of three panels, the left, middle, and right panels correspond to HA-UL24, nucleolin, and the merged image, respectively. Colocalization of the two signals is indicated by the yellow color in the merged panels.

FIG. 3.

Quantification of nucleolin distribution patterns in cells expressing HA-UL24 variants. COS-7 cells were transiently transfected with vectors expressing the HA-UL24 wild type or the substitution-containing forms indicated. Two days posttransfection, cells were costained for HA and nucleolin. The graph shows the percentage of cells expressing HA-UL24 that exhibited clear foci of nucleolin staining with little or no diffuse staining in the nucleus. Each result shown represents the average for three independent experiments, where more than 100 cells were analyzed for each mutation in each experiment. Error bars represent the standard errors of the means.

FIG. 4.

vUL24-eGFP. (A) Schematic representation of the insertion of an eGFP expression cassette in the UL24 ORF and the resulting BamHI fragments. The gray boxes represent the genes present in the BamHI Q fragment of the HSV-1 genome and the inserted eGFP expression cassette. The UL24 ORF is denoted by a white box. The black box between the CMV promoter and the eGFP gene represents the multiple cloning site from the plasmid pEGFP-N1. The top section represents the wild-type BamHI Q fragment and the insertion site of the eGFP cassette. The middle section represents the BamHI Q fragment following insertion. The bottom section shows the fragments obtained after BamHI digestion. (B) Genome analysis of vUL24-eGFPb and -c and vUL24-eGFPbResc. The ethidium bromide-stained agarose gel shows fragments generated following BamHI digestion of viral DNA and control plasmids. Plasmids contained either the wild-type BamHI Q fragment (pAG5) or the fragment containing the eGFP insertion (pBamH1QeGFP). Positions of the molecular mass markers are indicated to the left of the panel. (C) Southern blot analysis using a probe corresponding to the BamHI Q fragment in pBluescript II SK(+). The arrow to the right of the panel indicates the position of the fragment generated due to the insertion of the eGFP cassette. (D) Characterization of vUL24-eGFP viruses and vUL24-eGFPResc in a one-step growth curve. Results shown represent the average for two independent experiments done in duplicate. Error bars represent the standard errors of the means. (E) Plaque morphology was assessed for the different viruses on Vero cells 2 days postinfection grown at the indicated temperatures.

FIG. 5.

Characterization of vUL24-E99A/K101A and vUL24-G121A. (A) Western blot showing expression of UL24 in Vero cells infected with vUL24-E99A/K101A (a and b) (top panel). The blot was stripped and reblotted for the viral protein ICP8 as a control (bottom panel). (B) Western blot showing expression of UL24 in cells infected with vUL24-G121Aa and -b (top panel). The blot was stripped and reblotted for the viral protein ICP8 as a control (bottom panel). The positions of molecular mass markers are indicated to the left of each panel. Arrows to the right of the panels mark the position of the protein. (C) One-step growth analysis of vUL24-E99A/K101A (isolates a and b) and vUL24-G121A (isolates a and b) compared to the wild-type virus KOS and the UL24-deficient virus UL24X. (D) Analysis of plaque morphology for vUL24-E99A/K101Aa and -b or (E) for vUL24-G121Aa and -b on Vero cells at 2 days postinfection grown at the indicated temperatures.

FIG. 6.

Distribution pattern of nucleolin in Vero cells infected with UL24 mutants. (A) Confocal images of cells either mock infected or infected with the indicated virus. Cells were immunostained for nucleolin (green), and nuclei were stained with Draq5 (blue). Left-hand panels show nucleolin localization, center panels show stained nuclei, and merged images are shown in the right-hand panels. (B) Quantification of the distribution pattern for nucleolin in cells infected with the indicated UL24 mutants. Cells were either mock infected or infected at an MOI of 10 and stained for nucleolin at 18 hpi. Cells were analyzed by confocal microscopy, and the number of cells showing clear foci of nucleolin staining was scored. Each result shown represents the average for two independent experiments in which the localization of nucleolin in more than 150 cells was analyzed for each mutant virus. Error bars represent the standard errors of the means.