Article Figures & Data
Figures
- FIG. 1.
Diagram of the UL24 transcription unit. The relative positions and orientations of the UL24 and flanking ORFs are indicated by the fat arrows at the top of the figure. The six transcripts containing UL24 sequence that have been identified are illustrated below. The three long transcripts that terminate at the end of UL26 are expressed with leaky-late kinetics. The three short transcripts that terminate at the end of UL24 are expressed with early kinetics. Transcripts beginning at the second UL24 transcription start site are less abundant than those beginning at the other two start sites, which is reflected qualitatively by the relative thickness of the arrows.
- FIG. 2.
Western blot showing the effect of an ICP27-null mutation on the expression of UL24 protein in infected cells. Vero or V27 cells were either mock infected (lanes 1 and 4) or infected at an MOI of 10 with either KOS1.1 (lanes 2 and 5) or d27 (lanes 3 and 6) cells, and total cell lysates were harvested at 15 h p.i. and concentrated 10-fold. A 20-μl aliquot of each concentrated lysate was analyzed by Western blotting with antiserum raised against UL24, the position of which is indicated to the right of the top panel. In lanes 7 to 15, a dilution series of concentrated KOS1.1-infected cell lysate was loaded. The volumes (in microliters) are indicated above the panel. The membrane was subsequently stripped and incubated with an antibody directed against the HSV TK, the position of which is indicated to the right of the bottom panel.
- FIG. 3.
Effect of an ICP27-null mutation on the expression of UL24 transcripts in infected cells. Vero or V27 cells were either mock infected (lanes 1 and 4) or infected at an MOI of 10 with either KOS1.1 (lanes 2 and 5) or d27 (lanes 3 and 6) cells, and at 13 h p.i. total RNA was isolated. RNA was analyzed by Northern blot hybridization with a probe corresponding to the UL24 sequence. The position of the 1.4-kb short UL24 transcript is indicated by an arrow to the right of the panel. The position of the broad band corresponding to the 5.2-, 5.4-, and 5.6-kb-long UL24 transcripts is indicated by a bracket to the right of the panel.
- FIG. 4.
ICP27 affects the cytoplasmic versus nuclear distribution of the long, leaky-late UL24 transcripts. Vero cells (lanes 1 to 6) or V27 cells (lanes 7 to 12) were either mock infected (M) (lanes 1, 2, 7, and 8) or infected with KOS1.1 cells (lanes 3, 4, 9, and 10) or the ICP27-null virus d27 (lanes 5, 6, 11, and 12). (A) RNA was isolated from nuclear (N) and cytoplasmic (C) fractions at 13 h p.i., resolved on a denaturing formaldehyde agarose gel, and stained with ethidium bromide to allow for visualization of the major rRNA species. The positions of the various rRNAs are indicated by arrows to the left of the panel. RNA molecular mass markers (Ambion) were resolved in the first and last lanes of the gel (lanes m). The sizes of the markers are located to the right of the panel. (B) Nucleic acids from the agarose gel were analyzed by Northern blot hybridization for UL24 transcripts, the positions of which are indicated by arrows to the right of the panel. The bands corresponding to the long UL24 transcripts in the cytoplasmic fractions are distorted due to the comigration of the 28S rRNA.
- FIG. 5.
The 3′ UTR of the long UL24 transcripts is not sufficient to confer ICP27-dependent cytoplasmic localization. (A) Diagram illustrating the six 3′ coterminal transcripts that utilize the UL26 polyadenylation signal. The 5.6-, 5.4-, and 5.2-kb transcripts represent the long UL24 transcripts, the 4.2-kb transcripts originate from the UL25 promoter, the 2.4-kb transcripts originate from the UL26 promoter, and the 1.4-kb transcript corresponds to UL26.5. (B) The membrane analyzed in Fig. 4 was stripped and hybridized with a radioactive probe corresponding to the UL26.5 sequence. The positions of the UL25 (4.2-kb), UL26 (2.4-kb), and UL26.5 (1.4-kb) transcripts are indicated by arrows to the right of the panel. M, mock; K, KOS1.1; N, nuclear; C, cytoplasmic.
- FIG. 6.
Effect of ICP27 on the cytoplasmic localization of gB, VP16, and gC transcripts. (A) The membrane analyzed in Fig. 4 was stripped and hybridized with a radioactive probe corresponding to the gB sequence. The position of the gB transcripts is indicated to the right of the panel. (B) The membrane in Fig. 4 was stripped and hybridized with a VP16-specific probe (UL48). The positions of the 1.7-kb transcripts originating at the UL48 promoter, the 3.2-kb transcripts originating at the UL49 promoter, and the 3.8-kb transcripts originating at the UL49.5 promoter are indicated to the right of the panel. (C) The membrane in Fig. 4 was stripped and hybridized with a gC-specific probe. The position of the gC transcripts is indicated to the right of the panel. Abbreviations for all three panels: M, mock; K, KOS1.1; N, nuclear; C, cytoplasmic.
- FIG. 7.
Effect of ICP27 on the cytoplasmic localization of gB transcripts at earlier times in infection. (A) Vero cells were either mock infected (lanes 1, 2, 7, and 8) or infected with KOS1.1 (lanes 3, 4, 9, and 10) or the ICP27-null virus d27 (lanes 5, 6, 11, and 12). (A) At 5 h (lanes 1 to 6) or 10 h (lanes 7 to 12) p.i., total RNA was isolated from nuclear (N) and cytoplasmic (C) fractions, resolved on a denaturing formaldehyde agarose gel, and stained with ethidium bromide. The positions of precursor 45S and 32S rRNAs and fully processed 28S and 18S rRNAs are indicated by arrows to the left of the panel. RNA molecular mass markers are resolved in the first and last lanes of the gel (lanes m). The sizes of the markers are located to the right of the panel. (B) The membrane was hybridized with a gB-specific probe. The position of gB transcripts is indicated by an arrow to the right of the panel. M, mock; K, KOS1.1; N, nuclear; C, cytoplasmic.
- FIG. 8.
Effect of ICP27 on the cytoplasmic localization of various viral transcripts at earlier times in infection. (A) The membrane in Fig. 7 was stripped and hybridized with a UL26-specific probe. The positions of the UL26.5 (1.4-kb), UL26 (2.4-kb), UL25 (4.2-kb), and UL24 (5.6-kb) transcripts are indicated to the right of the panel. (B) The membrane in Fig. 7 was stripped and hybridized with a VP16 (UL48)-specific probe. The positions of the UL48 (1.7-kb), UL49 (3.2-kb), and UL49.5 (3.8-kb) transcripts are indicated to the right of the panel. (C) The membrane in Fig. 7 was stripped and hybridized with a UL42-specific probe. The position of the UL42 transcripts is indicated to the right of the panel. M, mock; K, KOS1.1; N, nuclear; C, cytoplasmic.
