Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA

Abstract

The virion host shutoff (vhs) protein encoded by the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (gamma) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the U(L)41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

Fig. 1.

vhs pulls down VP22 from infected cell lysates. One hour after mock infection or exposure to 10 pfu of HSV-1(F) per cell, confluent HeLa cell monolayers were incubated in methionine-free DMEM containing [³⁵S]methionine. A replicate set of cultures was maintained in medium containing 300 μg/ml PAA. The cells were harvested 18 h after infection and lysed in GST lysis buffer (see Materials and Methods). Precleared cell lysates were reacted with GST (lanes 1, 2, 5, and 6) or GST–VHS (lanes 3, 4, 7, and 8) for pull-down assays, resolved by PAGE, and transferred to nitrocellulose membrane. (A) Autoradiographic image of proteins pulled down by GST–VHS. (B) Immunoblotting with anti-VP16 and anti-VP22 antibodies.

Fig. 2.

The interaction between vhs and VP22 is mediated by VP16. HEK 293T cells were transiently transfected with plasmids encoding full-length VP22 or full-length VP16 either alone or in combination. Soluble cell extracts were prepared 48 h after transfection, reacted with glutathione–agarose beads bound to GST (lanes 4, 6, and 8) or GST–VHS (lanes 5, 7, and 9). The proteins bound to the beads were subjected to electrophoresis on a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with antibodies against VP16 (upper blot) or VP22 (lower blot). Immunoreactivity of VP22 (lanes 1 and 3) and VP16 (lanes 2 and 3) from a one-tenth volume of whole-cell lysates also is shown. cDNA, pcDNA 3.1(+).

Fig. 3.

Both VP16 and VP22 are required for the accumulation of vhs protein in transiently transfected cells. HEK 293T cells were transiently transfected with the listed plasmids, harvested 48 h after transfection, solubilized as described in Materials and Methods, and analyzed by immunoblotting for the accumulation of vhs (upper blot), VP22 (middle blot), and VP16 (lower blot). Immunoreactivity to actin (arrow) is shown as a loading control. The results are representative of at least five independent transfection experiments.

Fig. 4.

Wild-type vhs protein is not degraded by the proteasome pathway in the absence of VP22. HEK 293T cells were transiently transfected with the listed plasmids. Six hours before harvesting, the cells were incubated with mock medium (lanes 1 and 11) or medium containing DMSO (lanes 2, 5, 8, and 12) or 10 μM MG132 (lanes 3, 6, and 9) or 15 μM lactacystin (lanes 4, 7, and 10). The cells were solubilized as described in Materials and Methods and analyzed by immunoblotting for the accumulation of vhs (top blot), VP16 (middle blot), and VP22 (bottom blot).

Fig. 5.

VP22 and VP16 rescue the accumulation of a reporter gene coexpressed with vhs. HEK 293T cells were transiently transfected with the listed plasmids, harvested 48 h after transfection, solubilized as described in Materials and Methods, and analyzed by immunoblotting for the accumulation of (from top to bottom) vhs, EGFP, VP22, and VP16.

Fig. 6.

VP22 does not up-regulate the expression of vhs RNA. Cytoplasmic RNA was purified from HEK 293T cells harvested 48 h after transfection with the indicated plasmids. RNA (8 μg) was loaded onto denaturing formaldehyde gel, transferred onto a nylon membrane, and probed with ³²P-labeled fragment containing the entire coding sequence of vhs. The ethidium-bromide-stained rRNAs served as loading controls.

Fig. 7.

vhs stabilizes the interaction between VP22 and VP16. Plasmids encoding VP22 and full-length VP16 were transiently transfected in HEK 293T cells with (lanes 1 and 3) or without (lanes 2 and 4) the plasmid expressing wild-type vhs. Soluble cell extracts were prepared 48 h after transfection and incubated with mouse monoclonal anti-VP16 antibody (lanes 3 and 4). The proteins bound to the antibody were subjected to electrophoresis on a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with antibodies against VP16 and vhs (upper blot) or VP22 (lower blot). Immunoreactivity of VP22, VP16, and vhs (lanes 1 and 2) from a 1/10th volume of whole-cell lysates also is shown.