KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation

<div><p>TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi’s sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection.</p></div

Viral ORF57 inhibits eIF2α phosphorylation.

<p>(A) Schematic diagrams showing the governing process of SG formation and the possible mechanism by which ORF57 may prevent it. (B) ORF57 inhibits eIF2α phosphorylation. HeLa cells transfected with an empty (-) or ORF57 expressing (ORF57) vector were treated with arsenite. The phosphorylated eIF2α (p-eIF2α) was measured by Western blot analysis using a Ser 51 phosphor-specific eIF2α antibody. Total level of eIF2α was determined by a pan-eIF2α antibody. GAPDH served as a loading control. (C) Graphical representation of the relative amount of p-eIF2α (black) and eIF2α (white) in the panel B. The relative intensity of the protein band in each sample, after normalizing to GAPDH, was calculated over that of the arsenite-treated empty vector control. The error bar indicates mean ± SD (n = 3). (D-E) KSHV lytic infection in BCBL-1 cells does not increase eIF2α phosphorylation, but rather decrease arsenite-induced eIF2α phosphorylation. BCBL-1 cells with or without VA induction for lytic virus infection was treated with or without arsenite for 30 min before cell lysate preparation for Western blotting with corresponding antibodies (D). ORF57 was blotted as an indication for viral lytic induction. Relative amount of total eIF2α or p-eIF2α in each sample after normalizing to tubulin was measured and plotted in bar graphs for comparison (E), with each protein level in lane 1 (D) being set to 1. (F and G) Kinetic ORF57 production and p-eIF2α reduction in BCBL-1 cells with KHSV lytic infection. BCBL-1 cells induced with 1 mM VA for the indicated time for virus lytic infection were treated with arsenite for 30 min and then analyzed by Western blotting (F). The un-induced cells without arsenite treatment served as a negative control. S, short time exposure; L, longer time exposure. The relative amount of each protein in each sample after normalizing to tubulin was plotted over the time when the sample was collected (G), with the protein level in arsenite-treated cells without VA induction (0) being set to 100%. The error bar indicates mean ± SD (n = 2).</p

Knockdown of PKR expression increases production of KSHV virions.

<p>(A) Knockdown of PKR expression by a PKR-specific siRNA in iSLK-BAC16 cells by Western blot analysis. (B) Analysis of KSHV virus production after siRNA knockdown of PKR expression. KSHV replication in iSLK-BAC16 cells was induced by treatment with 1 mM sodium butyrate and 1 μg/ml doxycycline for 5 days. Supernatants obtained from the induced iSLK-BAC16 cells, which contain GFP-viruses, were used to infect HEK293 cells. The infected HEK293 cells were observed at 48 h infection for GFP expression as an indication of KSHV infection. (C) Virus-infected GFP-positive HEK293 cells were quantitated by FACS analysis. Each transfection/induction was performed in triplicate and three replicate infections were performed with each supernatant. Number of GFP-positive cells over the total number of cells in each infection was counted and expressed as a percentage (%). One representative of three infections is shown.</p

Viral ORF57 interacts with the RBM domain of PKR.

<p>(A) Schematic diagrams (not in scale) of PKR and its deletion mutants. RBM, dsRNA-binding motifs; PK domain, protein kinase domain. Numbers above each diagram represent amino acid positions in PKR protein (B) Mapping of ORF57-PKR interacting domains. HEK293 cell extract containing Myc-Flag-tagged full-length PKR or its deletion mutant ΔPK or ΔRBM was mixed with the cell extract containing untagged ORF57. The mixture was then digested with RNase A/T1 and immunoprecipitated with an anti-Flag antibody for PKR. The proteins in the pulldown were detected by Western blot using anti-ORF57 or anti-Flag for PKR. (C) ORF57 interacts more efficiently with phosphorylated PKR. HEK293 cells expressing full-length PKR-Myc-Flag were treated with arsenite for 30 min. Total cell extract was mixed with another HEK293 cell extract containing ORF57-Flag and digested with RNase A/T1. The PKR-ORF57 complex was pulled down with anti-Myc beads and blotted by anti-Flag antibody for total PKR, anti-phosphorylated PKR (Thr 451) for p-PKR and anti-ORF57 for ORF57.</p

KSHV latent infection is permissive for SG formation while the viral lytic infection with ORF57 expression is refractory.

<p>(A) Outlines of KSHV reactivation and stress granule induction in BCBL-1 or Bac36 cells. Circles, culture dishes; dotted squares, cells grown on cover slips. (B and C) Induction of SG by arsenite in KSHV-infected BCBL-1 and Bac36 cells. KSHV lytic infection in BCBL-1 was induced by valproic acid (VA, 1 mM) (B) and in Bac36 cells harboring a wild-type or ORF57-null KSHV genome (wt or Δ57) was induced by sodium butyrate (Bu, 3 mM) (C). The cells with or without VA or Bu induction for 24 h were further treated with 0.5 mM arsenite for 30 min and then immunostained for the SG-specific marker TIA-1 and viral lytic protein ORF57 (BCBL-1 cells and Bac36 wt cells) or RTA (Bac36 Δ57 cells). Bac36 cells with ORF57 or RTA expression (C) are separated from the cells without ORF57 or RTA expression by dashed white borderlines. The nuclei were counterstained with Hoechst dye. Scale bar = 10 μm. (D and E) Proportion of BCBL-1 and Bac36 cells with SG formation before and after virus lytic reactivation. Total of 50 cells in each group, ORF57-positive or RTA-positive (for Δ57 Bac36 cells) cells vs ORF57-negative or RTA-negative (for Δ57 Bac36 cells) cells, were counted in each experiment. The error bars represent SD from three independent experiments. **P<0.01 in Chi-squared test.</p

ORF57 interacts with PACT via its RBM1 and RBM2 motifs.

<p>(A) Schematic diagrams (not in scale) of full-length (FL) wt PACT and its deletion mutants. PACT contains two RNA-binding motifs (RBM1 and RBM2) and a PKR-activation domain (PAD). Numbers indicate the positions of amino acid (aa) residues for each domain in PACT. A series of PACT deletion mutants either with Δ1 missing RBM1 (aa 35–99), Δ2 missing RBM2 (aa 127–192), Δ3 missing PAD (aa 240–313) or double mutant Δ1,2 missing both RBM1 and RBM2) (aa 35–99 and aa 127–192) were generated by overlapping PCR. (B-D) Mapping of ORF57-PACT interacting domains. HEK293 cell extract containing ectopically-expressed Myc-Flag-PACT or its deletion mutant was mixed with the cell lysate containing untagged ORF57. The mixture was digested with RNase A/T1 and subjected to co-IP either with anti-Flag M2-coated beads (B, D) or with polyclonal rabbit anti-ORF57 antibody-coated beads (C). The proteins in the co-IP complex were detected by Western blot with the corresponding antibodies.</p

ORF57 inhibits autophosphorylation of PKR by blocking its interaction with Poly I:C, but does not directly affect elF2α phosphorylation.

<p>(A) Schematic diagrams of PKR activation. PKR binds to dsRNA poly I:C leading to its dimerization and autophosphorylation. The phosphorylated PKR at the kinase domain catalyzes eIF2α phosphorylation. (B) ORF57 interacts with PKR and prevents PKR from binding to poly I:C. Purified PKR-Myc-Flag or its mutant ΔRBM (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006677#ppat.1006677.g007" target="_blank">Fig 7A</a>) immobilized on Myc beads was incubated with ORF57 or BSA for 15 min at room temperature before addition of ³²P-poly I:C. Bar graphs with means ± SD (n = 4, each in triplicate) show the binding of ³²P-poly I:C to PKR or its ΔRBM mutant in the presence of ORF57 or BSA, quantified by scintillation count. Flag peptide addition to Myc beads served as a negative control for the Myc beads only (no immobilized PKR protein). (C) ORF57 inhibits PKR autophosphorylation. Purified PKR-Myc-Flag on Myc beads was used in the assay in the presence of [γ-³²P-ATP], with or without poly I:C or with poly I:C plus ORF57 or BSA. The incorporated ³²P into PKR due to autophosphorylation induced by poly I:C binding was measured by autoradiography of a SDS-PAGE gel after normalized to BSA control. The bar graph with means ± SD is derived from three independent experiments, with a representative SDS-PAGE gel image shown below the bar graph. (D) ORF57 doesn’t directly affect eIF2α phosphorylation. Inactive or arsenite-activated PKR-Myc-Flag immobilized on Myc beads was mixed with ORF57 or BSA protein and followed by addition of GST-eIF2α. An in vitro kinase reaction in the presence of [γ-³²P-ATP] was carried out to phosphorylate GST-eIF2α. Incorporated ³²P into GST-eIF2α was measured using SDS-PAGE and followed by autoradiography. The bar graph with means ± SD is derived from three independent experiments, with a representative SDS-PAGE gel image shown below the bar graph.</p

KSHV ORF37 (vSOX) inhibits arsenite-induced SG formation, but KSHV vIRF-2 does not.

<p>(A-B) KSHV ORF37 (vSOX) protein disrupts SG formation in HEK293 cells. HEK293 cells transfected with a vSOX-FLAG expression vector for 24 h were induced by arsenite for SG formation, fixed and stained with an anti-TIA-1 antibody (red) and an anti-FLAG antibody (green) for vSOX expression. The nuclei were counterstained with Hoechst dye (A). The cell lysates were blotted by an anti-FLAG antibody for vSOX protein (B). (C-D) KSHV vIRF2 does not inhibit SG formation and PKR phosphorylation. HeLa cells transfected for 24 h with an empty vector or a vector expressing KSHV vIRF2 or ORF57 were treated with arsenite to induce SG formation. Part of the transfected cells were used for IFA staining of SG-specific TIA-1 (red) in combination with anti-FLAG staining of vIRF2-Flag (green) (C) and other part of the cells were used for Western blotting (D). The nuclei were counterstained with Hoechst stain (C). Phosphorylated PKR (p-PKR) or eIF2α (p-eIF2α) in the cell lysate with or without arsenite treatment was blotted using a phosphor-specific PKR or eIF2α antibody and total level of PKR or eIF2α protein was blotted by an anti-PKR or anti-eIF2α antibody. The β-actin served as a loading control. Bar = 10 μm (A and C).</p

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

<div><p>HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16⁺ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of ⁹¹QYNK⁹⁴ to ⁹¹PSFW⁹⁴ displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.</p> </div

Viral ORF57 expression is required to suppress the formation of SG during KSHV lytic infection and under arsenite stress.

<p>(A) ORF57 expression is required to inhibit the formation of SG during KSHV lytic infection reactivated by RTA. Bac36 cells (wt or Δ57) were transfected either with a vector control (control) or with an RTA expression vector (RTA activation) to induce lytic KSHV infection. To observe SG, the cells were stained for TIA-1 (red) along with RTA (green) by each specific antibody. A positive control for SG was also set by arsenite treatment on Bac36 wt cells transfected with an empty vector. The nuclei were counterstained with Hoechst stain. Scale bar = 10 μm. Bar graph below the images shows the cells forming SG before and after virus lytic reactivation through RTA expression. HEK293 cells transfected with an empty or RTA expressing vector served as a negative control. Total of 100 cells in each group were counted in each experiment. The error bars represent mean ± SD (n = 3). **P<0.01 in Chi-squared test. (B) ORF57 alone is sufficient to inhibit SG formation. HeLa cells transfected with an ORF57-Flag (pVM7) expressing vector or an empty vector (control) for 24 h were induced with 0.5 mM arsenite for 30 min for SG formation. The cells were stained for ORF57 (green), SG-specific TIA-1 (red) and PABPC1 (white) by each corresponding antibody. The nuclei were counterstained with Hoechst stain. Scale bar = 10 μm. (C) The ORF57 N-terminal NLS is required to inhibit SG formation. HeLa cells were transfected either with an empty vector or a vector expressing ORF57-Flag (ORF57 wt), ORF57 mtNLS2+3-Flag (ORF57 mt) or ORF59-Flag (a viral DNA replication processivity factor) for 24 h and then induced by arsenite for 30 min for SG formation. Mouse anti-Flag antibody served to detect ORF57 wt, ORF57 mt and ORF59. TIA-1 antibody was used to probe SG. Scale bar = 10 μm. Bar graphs on the right are number of the cells with SG (upper) or number of SG per cell (lower) from at least 100 cells in each group. The error bar indicates mean± SD. **P<0.01 in Chi-squared test. (D) Relative amount of TIA-1 in insoluble pellets derived from HeLa cells with or without expression of functional ORF57. HeLa cells transfected with an ORF57 wt or ORF57 mt expressing vector or an empty Flag control vector for 24 h were either untreated (-) or treated (+) with 0.5 mM arsenite for 30 min to induce SG and then lysed in a sample buffer. The lysed samples were centrifuged at 15800 x g to separate soluble from insoluble fractions. The insoluble pellets containing SG were dissolved in SDS sample buffer for TIA-1 immunoblotting (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006677#ppat.1006677.s003" target="_blank">S3B Fig</a>). The relative amount of detectable TIA-1 in each sample, after normalized to tubulin loading control, was calculated over the amount of TIA-1 in arsenite-treated, Flag control pellets. **P<0.01 in student <i>t</i>-test. (E) Kinetic profile of TIA-1 in the insoluble pellets in the presence of an ORF57-expressing vector or an empty vector over the indicated time of arsenite treatment. Similar to Fig 2D, the insoluble pellets containing SG in HeLa cells with ORF57 (ORF57) or with an empty vector (control) were immunoblotted for the relative amount of TIA-1 in each time point (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006677#ppat.1006677.s003" target="_blank">S3C Fig</a>) and calculated as described above.</p