Kaposi's sarcoma-associated herpesvirus ORF57 protein protects viral transcripts from specific nuclear RNA decay pathways by preventing hMTR4 recruitment

Abstract

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5' RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.

Fig 1. ORF57 expression stabilizes PANΔENE in pulse-chase assays upon PPD inactivation.

(A, C, E & G) Representative northern blots of transcription pulse-chase assay in 293A-TOA cells transfected with an empty vector or ORF57 expression plasmid and with a non-targeting control siRNA (A) or a two-siRNA pool targeting PAPα/γ (C), PABPN1 (E), or ZFC3H1 (G). 7SK serves as loading control. The “-” lane was harvested prior to dox removal and the time 0 sample was taken two hours after dox removal immediately prior to re-introduction of dox. (B, D, F & H) Decays curves of biological replicates of the transcription pulse-chase assays; each point is a mean value with standard deviation (n = 3). Quantification was performed by normalizing PANΔENE values to 7SK. The t = 0 sample was set to 100% and other values were calculated relative to this sample. For each graph in Figs 1 and 2, the siCtrl/Vector data are re-plotted as a reference.

Fig 2. ORF57 acts on an ARS2-mediated RNA decay pathway.

(A, C, E & G) Representative northern blots of transcription pulse-chase assay in cells expressing an empty vector or ORF57 and transfected with a two-siRNA pool targeting ZCCHCH8 (A), ARS2 (C), PAPα/γ and ARS2 combined (E) or hMTR4 (G). 7SK serves as loading control. (B, D, F & H) Decay curves of biological replicates of the transcription pulse-chase assays (n = 3). See Fig 1 for additional details.

Fig 3. Generation and characterization of iSLK-ΔORF57 cells.

(A) Schematic diagram of ORF57 locus and deletion strategy. ORF56 (gray) and ORF57 (green) share a poly(A) signal. A 33-bp deletion containing the ORF57 initiating ATG and three additional in-frame ATG codons was made to minimize effects on ORF56 expression. (B) Quantitative western blot showing ORF57 protein from induced iSLK-WT, iSLK-ΔORF57 or iSLK-ΔORF57 cells transduced with lentivirus expressing ORF57. Actin serves as loading control. (C) Flow cytometry analysis of HEK293 cells infected with supernatants from indicated cell lines. The x-axis shows forward scatter and the y-axis indicates GFP. Percentage of GFP positives is shown. (D) Relative viral DNA levels in iSLK-WT (green) and iSLK-ΔORF57 (black) cells after induction. Values were calculated relative to WT 0 hours post induction (hpi). (E) Time course and northern blot of ORF59, ORF47 and PAN RNA mRNA from iSLK-WT and iSLK-ΔORF57 cells. (F) Relative ORF50 mRNA levels in iSLK-WT (green) and iSLK-ΔORF57 (black) determined by qRT-PCR either not induced or 24 hours post lytic reactivation. Values were first normalized to β-actin and calculated relative to WT (n = 3). In this and all figures, the open circles represent values from each biological replicate. (G) Ten-minute 4SU pulse labelling of iSLK-WT (green) and iSLK-ΔORF57 (black) cells. 4SU-containing ORF59, PAN RNA, and 7SK levels were measured by qRT-PCR after selection. Quantification was done by setting the value of WT samples to 1 for each transcript (i.e. ORF59, PAN RNA and 7SK). ΔORF57 values were calculated relative to the corresponding WT for each of three biological replicates. 7SK shows that there is no change between WT and ΔORF57 samples. The “- 4SU (ORF59)” samples used RNA from the indicated reactivated cell lines but were not treated with 4SU. These samples show background of the assay and were calculated relative to ORF59 WT. Values are average and error bars are standard deviations (n = 3).

Fig 4. Inactivation of two RNA decay pathways restores viral RNA levels in iSLK-ΔORF57 cells.

(A-E) Representative northern blots and quantification of ORF59 mRNA from iSLK-WT and iSLK-ΔORF57 transfected with a non-targeting control siRNA or a two-siRNA pool targeting ARS2 or hMTR4 (A), PAPα/γ (B), PABPN1 (C), ZFC3H1 (D), or ARS2/PAPα/γ co-depletion (E). Total RNA was purified 3 days after siRNA transfection, except for panel B where PAPα/γ were knocked down for 4 days. In all cases, cells were harvested 24 hours post lytic reactivation. All experiments included 3 biological replicates (n = 3) except for panels A and E, where quantifications were done from 5 and 4 replicates, respectively. 7SK RNA or GAPDH serves as loading control as indicated. (F) Representative northern blot and quantification (n = 3) of PAN RNA from iSLK-WT and iSLK-ΔORF57 transfected with a non-targeting control siRNA or a two-siRNA pool targeting PAPα/γ, ARS2 or both simultaneously. 7SK serves as loading control. (G) Quick 4SU pulse in iSLK-WT (green) and iSLK-ΔORF57 (black) cells transfected with a two-siRNA pool targeting both PAPα/γ and ARS2. In all graphs, mean values are plotted with standard deviation. Quantification was determined by first normalizing values to the corresponding loading control. iSLK-WT cells treated with control siRNAs was set as 1 and the rest of the values were calculated relative to it. Statistical analyses were two-tailed unpaired Student’s t-tests (*p<0.05).

Fig 5. ORF57 reduces hMTR4 recruitment to viral transcripts.

(A-B) Native RIP with IgG, ARS2, ORF57 or hMTR4 antibodies using extracts made from induced (24 hpi) iSLK-WT (green) or iSLK-ΔORF57 (black) cells. (C-D) CLIP with IgG, ORF57 or hMTR4 antibodies using extracts from induced (24 hpi) iSLK-WT (green) or iSLK-ΔORF57 (black) cells. The “no UV” and IgG samples show background of the assay. Values are average and error bars are standard deviations (n = 3). (E) Representative in vitro RNA IP assay. Extracts from cells expressing either an empty vector or ORF57 were incubated with an in vitro transcribed, radioactively labeled PAN RNA probe either capped or uncapped as indicated. Extracts were immunoprecipitated using IgG, ORF57, ARS2 or hMTR4 antibodies. 10% of input is shown. Bottom panel shows quantification of IPs using extracts expressing ORF57 (green), empty vector (black), an uncapped probe +ORF57 (gray) or an uncapped probe -ORF57 (white) (n = 3 for capped samples; n = 2 for uncapped). All values were calculated relative to anti-ORF57 in the ORF57 expressing extract. Statistical analyses were performed using two-tailed unpaired Student’s t-tests (*p<0.05, **p<0.01).

Fig 6. Gene-specific effects of ALYREF overexpression on ORF57-dependent viral RNAs.

(A-B) Bar graphs showing results from qRT-PCR of ORF59 and PAN RNA (A), and ORF6, ORF8, and ORF9 (B) obtained from 293i-ORF57 (green) or 293i-ΔORF57 (black) cells. The cells were transfected with an empty vector (EV), or ALYREF overexpression construct (gray) as indicated. 293i-ΔORF57 cells were also subject to depletion of hMTR4 (blue), or ARS2/PAPα/γ co-depletion (orange). Lytic reactivation was achieved by transfecting cells with a plasmid expressing ORF50. Total RNA was harvested 36 hours post ORF50 transfection. Values are average and the error bars are standard deviations (n = 3). (C) Time course and representative northern blot of ORF6 from iSLK-WT, iSLK-ΔORF57 and iSLK-ΔORF57 cells transduced with a lentivirus expressing ORF57. 7SK serves as loading control. (D) Ten-minute 4SU pulse labeling in iSLK-WT (green) and iSLK-ΔORF57 (black) cells. ORF6 mRNA levels were measured by qRT-PCR. Quantification was performed as in Fig 3G. The “- 4SU” values were calculated relative to ORF6 WT. Values are average and error bars are standard deviations (n = 3).

Fig 7. Model of ORF57-mediated protection of KSHV transcripts.

(A) In the absence of ORF57, hMTR4 interacts with ARS2 and recruits the nuclear exosome to viral transcripts to degrade them. (B) When ORF57 is expressed, it directly binds to viral transcripts and recruits the export adaptor ALYREF. The ORF57 recruitment of ALYREF allows it to outcompete hMTR4 for ARS2 binding to protect viral transcripts from exosome-mediated degradation. (C) For ALYREF-independent, ORF57-dependent RNAs (e.g. ORF8 and ORF9), we speculate that ORF57 recruits an unknown cellular factor(s) (black square) which prevents hMTR4 recruitment to viral RNAs. See text for more details. For simplicity, PPD factors were omitted from the diagram.