Upstream-binding factor is sequestered into herpes simplex virus type 1 replication compartments

Abstract

Previous reports have shown that adenovirus recruits nucleolar protein upstream-binding factor (UBF) into adenovirus DNA replication centres. Here, we report that despite having a different mode of viral DNA replication, herpes simplex virus type 1 (HSV-1) also recruits UBF into viral DNA replication centres. Moreover, as with adenovirus, enhanced green fluorescent protein-tagged fusion proteins of UBF inhibit viral DNA replication. We propose that UBF is recruited to the replication compartments to aid replication of HSV-1 DNA. In addition, this is a further example of the role of nucleolar components in viral life cycles.

Fig. 1.

UBF is enriched in viral replication centres. (a) Cells infected with HSV-1 and probed with ICP8 antibodies at 7 h p.i. The location of UBF is shown in green, ICP8 protein is in red and the extent of the cell nucleus is revealed by staining with DAPI in blue. (b) Infected cells at 7 h p.i. (top row) and 24 h p.i. (bottom row). Arrows point to sites of extra nucleolar accumulation of UBF. The location of UBF is in green, B23.1 is in red and DAPI is in blue. All images are of a single focal plane approximately 0.3 μm in depth. Bar, 10 μm. (c) Location of a number of UBF–EGFP fusion proteins in HeLa cells at 7 h p.i. In each case, the plasmid indicated had been transfected into the cells approximately 15 h prior to infection. The EGFP fusion is shown in green, ICP8 is in red and DAPI is in blue. UBF–EGFP contains the full-length protein. (d) Schematic diagram of UBF. The drawing indicates the relative location of the 6 HMG boxes (numbered 1–6), the dimerization domain (marked with checked fill) and the transactivation domain (marked with diagonal lines). In addition, below the diagram the approximate locations of the C-terminal amino acids of the deletion mutants used in this study (full-length UBF is 764 aa) are indicated.

Fig. 2.

HSV-1 infection causes UBF to separate from RNA pol I and the major sites of RNA synthesis. HSV-1-infected cells at 7 h p.i., where the top row shows endogenous UBF (green), RNA pol I (red) and DAPI (blue). Similarly infected cells are shown in the middle row, endogenous UBF (green), RNA synthesis (detected by FU labelling for 20 min; red) and DAPI (blue) are shown. Similarly infected cells are also shown in the bottom row, where the location of FU (green), RNA pol I (red) and DAPI (blue) are shown. All images are of a single focal plane approximately 0.3 μm in depth. Bar, 10 μm.

Fig. 3.

The effects of HSV-1 infection on UBF and the effects of UBF fusion proteins on HSV-1 replication. In (a) the levels of total UBF and phosporylated UBF were assessed by Western blotting. On the left is a Western analysis of cells harvested at 7 and 24 h p.i., showing levels of total UBF (both isoforms) by using an affinity purified anti-UBF serum raised in sheep. The same samples were analysed for phosphorylated ser 388 UBF. Also shown is a control Western blot using antisera to actin as a loading control. (b) Shows the effect of expression of UBF–EGFP fusion proteins on HSV-1 origin-dependent DNA synthesis in the presence of the indicated UBF fusion proteins or pE9CT. The position of the replicated pS1 molecules are indicated by the arrow. (c) Shows the effects of the indicated plasmids on the ability of infectious HSV-1 DNA to generate plaques in a co-transfection assay.