A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export

Abstract

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

Keywords: 3′ UTR; KSHV; Nup98; ORF10; Rae1; herpesvirus; late gene; mRNA nuclear export.

Fig. 1

ORF10 induces poly(A)⁺ RNA accumulation in the nucleus. Poly(A)⁺ RNA was detected by in situ oligo(dT) hybridization. The poly(A)⁺ RNA signals of all cells (n>24) across four fields were quantified with ImageJ, and the Nu/Cy signal ratio was calculated and plotted as mean ± SD. (A) SLK cells were infected with MHV-68 for indicated hours, and the Nu/Cy signal ratio of poly(A)⁺ RNA was shown in (B). (C) SLK cells were mock infected (M) or infected with the wild-type MHV-68 (WT), the ORF10-null mutant (10S) or the revertant (10R) virus at an MOI of 3 for 15 hours and the Nu/Cy signal ratio of poly(A)⁺ RNA was shown in (D). (E) SLK cells were transfected with YFP-ORF10, ORF10-FLAG, or empty vectors, and the Nu/Cy signal ratio of poly(A)⁺ RNA was shown in (E). Scale bar: 20 μm.

Fig. 2

ORF10 interacts with Rae1, and further forms a complex with Nup98. (A) ORF10 interacts with Rae1 in co-IP. (B) Rae1 mediates the interaction between ORF10 and Nup98. (C) ORF10, Rae1 and Nup98 form a complex in sequential IP. (D) Localization of ORF10 during MHV-68 infection. SLK cells were infected with the FLAG-tagged ORF10 MHV-68 virus (MHV68-F10) and after 16 hours, cells were fixed and subjected to IFA with αFLAG and αNup98 antibody. (E) Nuclear envelope enrichment of ORF10 requires Rae1 and Nup98. SLK cells were transfected with control siRNA (siCtl), siRNA against Rae1 or Nup98 for 2 days, followed by infection with the MHV68-F10 virus, 16 hours later, cells were fixed and subjected to IFA with αFLAG antibody. Scale bar: 20 μm. See also Fig. S1.

Fig. 3

The interaction with Rae1 is required for ORF10 to induce nuclear accumulation of poly(A)⁺ RNA. (A) Increasing the expression of Rae1 reverts nuclear poly(A)⁺ RNA accumulation induced by ORF10. SLK cells were co-transfected with a fixed amount (100ng) of ORF10-FLAG and increasing amount of HA-Rae1, and then subjected to in situ oligo(dT) hybridization and IFA with αFLAG antibody. (B) The Nu/Cy signal ratio of poly(A)⁺ RNA in (A). ORF10-negative cells were quantified as mock. Signal in all cells across four fields was quantified with ImageJ, and data are represented as mean ± SD. (C) Site-specific mutagenesis of ORF10 identified the mutations at three sites that disrupt the interaction with Rae1. (D) Distribution of poly(A)⁺ RNA in cells expressing WT or loss-of-interaction mutants of ORF10 was examined by in situ oligo(dT) hybridization and ORF10 expression examined by IFA with αFLAG antibody. (E) The Nu/Cy signal ratio of poly(A)⁺ RNA in (D) determined as in (B). Scale bar: 20 μm. See also Fig. S2.

Fig.4

ORF10 inhibits gene expression through blocking mRNA export. (A) ORF10 inhibits GFP reporter expression. 293T cells were transfected with pEGFP-C1 and ORF10-FLAG or vector control, and two days later, GFP protein expression was visualized under fluorescence microscope. (B) ORF10 inhibits nuclear export of GFP mRNA. The Cy and Nu RNAs were purified from transfected cells in (A), and the GFP transcript was quantified by RT-qPCR. The Nu/Cy ratio was plotted as mean ± SD (n=3). (C) ORF10 induces hyperadenylation of nuclear GFP RNA. The Cy and Nu RNAs from transfected cells in (A) were subjected to northern blotting with the labeled GFP or GAPDH probe. (D) Rae1 knockdown reduces GFP reporter expression. (E) Quantification of the fluorescence intensity in (D). (F) Expression of indicated proteins was analyzed by western blotting. (G) The loss-of-interaction ORF10 mutants failed to reduce GFP expression. (H) Quantification of the fluorescence intensity in (G). All fluorescence data with error bars are plotted as mean ± SD (n=5). Scale bar: 100 μm.

Fig 5

ORF10 inhibits gene expression via the 3'UTR region. (A) ORF10 inhibit GFP expression from pEGFP-C1, but not from pEGFP-3'GAPDH or pEGFP-3'GH. (B) Quantification of the fluorescence intensity in (A). (C) The Cy and Nu RNAs were purified from transfected cells, and analyzed by northern blotting with the labeled GFP probe. Images from both short and long exposure are shown. (D) Impact of Rae1 knockdown on GFP reporter expression. (E) Quantification of the fluorescence intensity in (D). (F) Expression of indicated proteins was analyzed by western blotting. Scale bar: 100 μm. All data with error bars are plotted as mean ± SD (n=5).

Fig. 6

ORF10 inhibits mRNA export of cellular genes. (A) The Cy and Nu RNAs were purified from transfected cells and quantified for the transcript level of RUNX1 and RBL2 by RT-qPCR. The Nu/Cy ratio was plotted as mean ± SD (n=3). (B) Expression of RUNX1 and RBL2 proteins in transfected cells was analyzed by western blotting. (C) An inducible cell line of ORF10 was transfected with Rae1 siRNA or control siRNA and then treated with tetracycline to induce ORF10 expression. Two days later, expression of RBL2 and RUNX1 were analyzed by western blotting. (D) The GFP constructs fused with the 3'UTR of RBL2 (pEGFP-3'RBL2) or RUNX1 (pEGFP-3'RUNX1) are sensitive to ORF10 inhibition. (E) Quantification of the fluorescence intensity in (D). (F) The Nu/Cy ratio of GFP RNA in cells of (D) is plotted as mean ± SD (n=3). Scale bar: 100 μm. See also Figs. S3, S4 and S5, Tables S2, S3 and S4.

Fig. 7

Efficient KSHV late gene expression requires ORF10 and its interaction with Rae1. (A) KSHV-latently infected iSLK cells, iSLK-WT, iSLK-10S and iSLK-10R, were transfected with Rae1 siRNA or control siRNA and then treated with doxycycline and sodium butyrate to induce viral lytic replication. The supernatants were collected to quantify infectious virions. Data are plotted as mean ± SD (n=3). (B) The lysates were harvested from cells in (A) for analyzing viral and cellular protein expression. (C) Infectious virion production from KSHV WT or ORF10 mutants, including 10S, two clones of 10m5, and two clones of 10m10. (D) Viral and cellular protein expression. (E) Genome-wide analysis of viral lytic gene expression for WT and ORF10 mutants. Total RNAs were purified from iSLK-WT, −10S, −10m5#1 and 10m10#1 cells, and subjected to RNA sequencing. The relative viral gene expression to the WT virus was plotted as means ± SD (n=3). See also Fig S6, Tables S5 and S6.