Host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm

Abstract

Lytic infection with the two human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), leads to significant depletion of the cellular transcriptome. This host shutoff phenotype is driven by the conserved herpesviral alkaline exonuclease, termed SOX in KSHV and BGLF5 in EBV, which in gammaherpesviruses has evolved the genetically separable ability to target cellular mRNA. We now show that host shutoff is also a prominent consequence of murine gammaherpesvirus 68 (MHV68) infection, which is widely used as a model system to study pathogenesis of these viruses in vivo. The effector of MHV68-induced host shutoff is its SOX homolog, here termed muSOX. There is remarkable functional conservation of muSOX host shutoff activities with those of KSHV SOX, including the recently described ability of SOX to induce mRNA hyperadenylation in the nucleus as well as cause nuclear relocalization of the poly(A) binding protein. SOX and muSOX localize to both the nucleus and cytoplasm of infected cells. Using spatially restricted variants of these proteins, we go on to demonstrate that all known host shutoff-related activities of SOX and muSOX are orchestrated exclusively from the cytoplasm. These results have important mechanistic implications for how SOX and muSOX target nascent cellular transcripts in the nucleus. Furthermore, our findings establish MHV68 as a new, genetically tractable model to study host shutoff.

FIG. 1.

MHV68 promotes host shutoff. 3T3 cells were either mock infected or infected with MHV68 at a multiplicity of infection of 10. (A) At the indicated times postinfection, cells were pulse-labeled with [³⁵S]-labeled Cys/Met and lysates were resolved by SDS-PAGE. The gel was then dried and visualized by autoradiography. (B) RNA was harvested from uninfected cells or cells infected for the indicated times and Northern blotted with GAPDH and 18S (loading control) probes. Quantification (normalized to 18S levels) is shown, with the level of GAPDH mRNA in the uninfected sample set to 1.0. PAA indicates the sample infected and cultured in the presence of 200 μg/ml PAA. (C) Lysates were harvested from either uninfected cells or cells infected with MHV68 for the indicated times and Western blotted with anti-muSOX antibodies. All figures are representative of three independent experiments. hpi, hours postinfection; α-muSOX, anti-muSOX.

FIG. 2.

muSOX enhances mRNA degradation. (A) 293T cells were transfected with the indicated plasmids for 24 h. Total RNA was then isolated and analyzed by Northern blotting with GFP and 18S probes. Quantification (normalized to 18S levels) is shown, with the level of GFP mRNA in the absence of muSOX set to 1.0. (B) Cells were transfected as described above, and the GFP mRNA half-life was calculated by qPCR at different time points post-ActD (2 μg/ml) treatment. (C) IVT muSOX or GFP (as a negative control) was incubated for 1 or 15 min with linear plasmid DNA in degradation assay buffer at 37°C. The DNA was then extracted, resolved by agarose gel electrophoresis, and visualized by ethidium bromide staining. (D) 293T cells were transfected with empty vector or with pCDEF3-GFP alone or together with pCDEF3-muSOX. With the exception of the control (−MNase), all samples were treated with micrococcal nuclease prior to cell lysis to remove extracellular nucleic acids. Samples were then divided in half and harvested for either total cellular DNA or RNA. GFP DNA and mRNA levels were then calculated via qPCR. (E and F) Cells were transfected as described for panel A. The endogenous GAPDH (E) or β-actin (F) mRNA half-life was then calculated via quantification of Northern blots (normalized to 18S) at different time points post-ActD treatment. Errors bars show the standard error between samples. All graphs represent a compilation of at least three independent experiments. t_1/2, half-life.

FIG. 3.

muSOX is necessary for viral replication and host shutoff during infection. (A) EcoRI and PvuII digests of WT MHV68, MHV68ΔmuSOX, and MHV68ΔmuSOX-MR. In the PvuII digest, asterisks represent cleavage products generated as a result of introducing an additional PvuII site upon deletion of the muSOX gene. (B) Growth curve comparing viral titer following transfection of 3T3 cells with WT, MHV68ΔmuSOX, and MHV68ΔmuSOX-MR BAC DNA. Limit of detection is 10 PFU/ml. Data are a compilation of three independent replicates with the WT and deletion BACs and two independent replicates with the MR BAC. (C) The indicated BAC DNAs were transfected into BHK cells in the presence of 200 μg/ml PAA. BAC-derived GFP mRNA abundance was determined by qPCR at different time points posttreatment with 5 μg/ml ActD. Errors bars show the standard error between samples from three independent experiments. t_1/2, half-life.

FIG. 4.

SOX homologs of gamma-HVs localize to both the nucleus and cytoplasm. (A) 293T cells were transfected with empty vector or HA-tagged SOX (KSHV), muSOX (MHV68), BGLF5 (EBV), and AE (HSV-1). Twenty-four hours posttransfection, cells were subjected to IFA with anti-HA antibodies. (B) HFF cells were either mock infected or infected with KSHV and lytically reactivated with an adenoviral vector expressing RTA. At the indicated times, samples were subjected to IFA with anti-ORF45 (KSHV lytic marker) (center panels) and anti-SOX (bottom panels) antibodies. (C) 3T3 cells were either mock infected or infected with BAC-derived MHV68. At the indicated times, samples were subjected to IFA with anti-muSOX antibodies. GFP is encoded by the BAC and serves as a marker of infection. Bottom panels show a magnified view of representative muSOX-expressing cells at each time point. All samples were costained with DAPI to visualize nuclei. α, anti.

FIG. 5.

Host shutoff is orchestrated from the cytoplasm. (A) 293T cells were transfected with the indicated plasmid expressing HA-tagged muSOX or muSOX fused to a WT or mutant NRS, as diagramed. Localization of each muSOX protein was visualized by IFA with anti-HA antibodies. (B) The host shutoff activity of each muSOX variant was assessed by cotransfection of a GFP reporter with either empty vector or the indicated muSOX-expressing plasmid. At 24 h posttransfection, cells were subjected to IFA with anti-HA antibodies. (C) 293T cells were transfected with increasing amounts of the indicated muSOX constructs (100 to 300 ng). Total RNA was harvested 24 h posttransfection and Northern blotted with GFP and 18S probes. Protein levels were assessed by Western blotting with anti-HA antibodies. (D) Linearized pCDEF3 plasmid was incubated with aliquots of the indicated IVT protein for 1 or 15 min in degradation assay buffer at 37°C. The DNA was then extracted, resolved by agarose gel electrophoresis, and visualized by ethidium bromide staining. α, anti; Vec, vector.

FIG. 6.

Partial relocalization of HSV-1 AE into the cytoplasm is not sufficient to induce mRNA turnover. (A) 293T cells were transfected with plasmids expressing either WT HSV-1 AE or AE-NLSmut for 24 h and then subjected to IFA with anti-HA antibodies. DAPI staining was used to visualize nuclei. (B) 293T cells were transfected with a GFP reporter plasmid alone or together with the indicated AE construct for 24 h and then subjected to IFA with anti-HA (α-HA) antibodies. (C) 293T cells were transfected with increasing quantities of the indicated plasmids (100 to 300 ng). Total RNA was harvested 24 h posttransfection and Northern blotted with GFP and 18S probes. AE and muSOX levels were assessed by Western blotting with anti-HA antibodies. (D) The linearized pCDEF3 plasmid was incubated with aliquots of the indicated IVT protein for 1 or 15 min in degradation assay buffer at 37°C. The DNA was then extracted, resolved by agarose gel electrophoresis, and visualized by ethidium bromide staining. Vec, vector.

FIG. 7.

The cytoplasmic fractions of muSOX and SOX drive mRNA hyperadenylation and PABPC nuclear import. (A) 293T cells were transfected with the indicated plasmids for 24 h and then subjected to oligo(dT) in situ hybridization (top panels), followed by staining with anti-SOX MAbs (SOX samples), anti-HA antibodies (muSOX samples), or both anti-HA and anti-SOX antibodies (vector sample) (center panels). The bottom panels show a merge of the in situ and IFA staining. The far right panels show a magnified view of a field of muSOX-transfected cells; the bottom right panel of this column shows a merge of the in situ signal and DAPI-stained nuclei. Arrows identify select muSOX-expressing cells. (B) 293T cells were transfected with a GFP reporter plasmid alone or together with the indicated muSOX- or SOX-expressing plasmid. At 24 h posttransfection, samples were treated with 5 ng/ml leptomycin B for 6 h to stabilize hyperadenylated RNAs, which are detected as the slower-migrating population (28). Total RNA was then harvested and Northern blotted with GFP and 18S probes. (C) A fraction of each RNA sample from that shown in panel B was incubated with oligo(dT) to bind poly(A) tails and then subjected to RNase H digestion to deadenylate the mRNAs prior to agarose-formaldehyde gel electrophoresis and Northern blotting. (D) 293T cells were transfected with empty vector or with a plasmid expressing the indicated muSOX fusion protein for 24 h and then subjected to double-label IFA with polyclonal anti-PABPC antibodies (center panels) and anti-HA or anti-SOX antibodies (top panels). The bottom panel shows a merge of the PABPC-stained and DAPI-stained nuclei. Arrows point to representative cells exhibiting PABPC nuclear import. α, anti.