Effects of US11 on the stability and immune signaling activity of duplex RNA formed by annealing of partially complementary reporter mRNAs.

A) Schematic of dsRNA generation. MCherry expression plasmids with a fragment of the SRPRα ORF (canine signal recognition particle receptor subunit alpha) inserted in the 3’UTR in the forward (fwd) or reverse (rev) orientation. Co-transfection of both plasmids results in formation of dsRNAs with 5’ and 3’ overhangs. B) HeLa cells were transfected with fwd and rev together (fwd/rev) along with either pEGFP or pEGFP-US11 for 20 hours. As a control, cells in separate dishes were transfected with either fwd/pEGFP or rev/pEGFP and combined during Trizol lysis (fwd)+(rev). Total RNA was extracted, digested by DNaseI and RNaseA/T1 (high salt conditions) or DNaseI, RNaseIII and RNaseA/T1 and analyzed by Northern blot with a probe specific for the inserted SRPRα fragment. C) Cotransfection of fwd/rev and pEGFP or pEGFP-US11, respectively. Transfected cells were treated with actinomycin D for 0, 2 or 4 hours prior to RNA extraction and processed as in (B). D) Quantification of the full-length dsRNA SRPRα signal from 3 independent experiments. P-value for pEGFP/4h vs. 0h is 0.010, 4h/pEGFP-US11 is ns/nonsignificant. E) HeLa cells were transfected with fwd or fwd/rev and pcDNA, pEGFP or pEGFP-US11, respectively. After 20 hours cells were treated with actinomycinD for 4 hours. Whole-cell lysates were analyzed by Western blot with the indicated antibodies. Results shown are representative of 3 independent experiments.</p

Accumulation of dsRNA in cells infected with vhs-deficient viruses.

A) HFF and HeLa cells were infected for 12 hours with HSV-1 (KOS37) WT, Vhs-, US11-, Vhs-US11-, IEUS11 and IEUS11 Vhs- at an MOI of 10. Total RNA was extracted and 1μg in 1μl was spotted onto a nylon membrane, UV-crosslinked and processed like a Western blot. DsRNA was detected by staining with the dsRNA-specific antibody J2. B) Quantification of dsRNA in mock-infected HeLa and HFF cells from 5 independent experiments, * pC) Quantification of dsRNA signal in HSV- and mock-infected HFF cells from 6 independent experiments, * pD) Quantification of dsRNA signal in HSV- and mock-infected HeLa cells from 3 independent experiments, * p<0.05.</p

Effects of vhs on the levels of partially complementary viral transcripts and dsRNA levels.

A and B) Diagram of the UL23/UL24 (A) and UL30/UL31 (B) regions of the HSV-1 genome, with the transcripts detected by Tombacz et al. 2017 and earlier studies depicted. The probes used for northern blots analysis are diagrammed. Coordinates are from the HSV-1 strain 17 reference genome (X14112). C and D) HeLa cells were infected with HSV-1 (KOS) WT and Vhs- for 6 or 12 hours at an MOI of 10. Equal amounts of total RNA were analyzed by Northern blot with probes specific for either the UL23 (C) or UL24 (D) transcripts. Results shown are representative of 3 independent experiments. E) Detection of UL23/UL24 duplex RNA. HeLa cells were infected with HSV-1 (KOS) WT and Vhs- for 12 hours at an MOI of 10. Total RNA was extracted and digested with RNaseA/T1 (high salt buffer) or RNaseIII followed by RNaseA/T1. Undigested input (1/10) and digested samples were analyzed by Northern blot with a probe specific for the predicted overlap between the UL23 and UL24 transcripts. F) Detection of UL30/UL31 duplexRNA. The experiment was performed as in E) but with a probe specific for the predicted overlap between UL30 and UL31 transcripts. Results shown in E) and F) are representative of 4 independent experiments each.</p

Virion-derived vhs accelerates decay of ectopically expressed dsRNA and concomitantly reverses PKR activation.

A) HeLa cells were transfected with fwd/rev and either pEGFP or pEGFP-US11 for 20 hours. Cells were mock infected or infected with HSV-1 (KOS) and HSV-2 WT and Vhs- at MOI 20 for 4 hours with continuous actD treatment. Total RNA was extracted, digested by DNaseI and RNaseA/T1 (high salt conditions) and analyzed by Northern blot with a probe specific for the inserted SRPRα fragment. B) Quantification of full-length SRPRα dsRNA from 3 (HSV-2) or 6 (HSV-1) independent experiments. P-values vs. mock: * p-: ns (HSV-2/pEGFP), pC) Cells were transfected with fwd/rev and pEGFP-US11 for 20 hours and infected as described in A). Whole cell lysates were analyzed by Western blot with the indicated antibodies. Results shown are representative of 3 independent experiments. D) Virion-derived vhs reverses PKR activation by preformed dsRNA. Cells were transfected with fwd/rev and pEGFP for 20 hours and infected as described in A). Whole cell lysates were analyzed by Western blot with the indicated antibodies. E) Quantification of P-PKR levels from 3 independent experiments. P-values vs. fwd/rev/mock: *p- p<0.0001, HSV-1 WT vs. Vhs- p<0.02.</p

Reduction of preformed dsRNA by virion-derived vhs is sensitive to hippuristanol.

A) HeLa cells were transfected with fwd or fwd/rev for 20 hours. Cells were then mock treated or treated with RNaseIII or RNaseA/T1 (high salt buffer) and analyzed by immunofluorescence with the dsRNA-specific antibody J2 (green). B) HeLa cells were transfected with fwd/rev for 20 hours and mock infected or infected with HSV-1 (KOS) and HSV-2 WT and Vhs- at an MOI of 20 for 5 hours in the continuous presence of actinomycin D. After immunofluorescence staining with the J2 antibody the ratio of J2 positive cells was determined by analyzing more than 100 mCherry-positive cells per coverslip. The ratio of J2 positive to mCherry positive cells in the mock infected sample was set as 100%. The graph represents the average of 4 independent experiments. P-value vs. mock: * pC) Hippuristanol (hip) antagonizes the dsRNA-destabilizing effect of vhs. The experiment was performed as described in B) with addition of vehicle control (0nM) or hip at 80 or 160nM during the course of infection. The graph represents the average of 4 independent experiments. P-values WT 0nM hip vs. mock 0nM hip: * p<0.005, ** p<0.0005; p-value 160nM hip vs. 0nM hip: p<0.05 (HSV-2), p<0.005 (HSV-1).</p

Vhs and US11 make independent and complementary contributions to PKR suppression during HSV infection.

HFF and HeLa cells were infected for 12 hours with HSV-1 (KOS37) WT, Vhs-, US11-, Vhs-US11-, IEUS11 and IEUS11 Vhs- at an MOI of 10. A) Whole-cell lysates were analyzed by Western blot with the indicated antibodies. Note that the Vhs antibody used cross-reacts with a co-migrating cellular protein present in HFF cells. B) Quantification of the P-PKR signal in HFF and HeLa cells. Data are from 4 independent experiments. The P-PKR signal in cells infected with Vhs-US11- was set at 100%.</p