A viral RNA structural element alters host recognition of nonself RNA

Abstract

Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2'-O methylation of the 5' cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5' cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5' untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5'-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

Figure 1. VEEV TC83 but not TRD is restricted by Ifit1

(A) Flow cytometry contour plots showing infection of TC83 in IFNβ-treated HeLa cells transduced with shRNA against a scrambled non-silencing control (NSC), human STAT2, or human IFIT1 (shNSC vs. shIFIT1 P < 0.003). One representative experiment of four is shown. This phenotype was confirmed with a second shRNA against IFIT1. (B) Survival of 4 week-old WT (n = 10) and Ifit1^−/− (n = 10) mice after s.c. infection with 10⁶ FFU of TC83. Results are pooled from three independent experiments. P values for survival were calculated using the Log-rank test. (C-D) Viral burden in 4 week-old WT or Ifit1^−/− mice infected s.c. with 10⁶ FFU of TC83, as measured in (C) draining popliteal lymph node (DLN) and (D) brain. Results are from 5 to 9 mice per tissue. Asterisks indicate statistically significant differences, as judged by an unpaired t test (** P < 0.005, *** P < 0.0001). Dashed lines indicate the limit of detection of the assay. (E) WT and Ifit1^−/− MEFs were pre-treated with 10 IU/ml of IFNβ for 12 hours or left untreated, and then infected with TC83 (MOI of 0.1). Supernatants were harvested for virus titration (WT versus Ifit1^−/− P > 0.2; WT + IFNβ versus Ifit1^−/− + IFN, 12, 24, 36 hours post-infection, P < 0.03). Each point represents the average of three experiments performed in triplicate, and error bars represent the standard error of the mean (SEM). P values were determined by an unpaired t test. (F) Survival curves of 8 week-old WT (n = 10) and Ifit1^−/− (n = 24) mice after s.c. infection with 50 PFU of TRD. Results are pooled from two independent experiments. P values for survival were calculated using the Log-rank test.

Figure 2. Mutations in the 5′-UTR determine Ifit1 sensitivity in vitro. (A and B)

Growth kinetics of (A3)VEE/SINV and (G3)VEE/SINV viruses in WT and Ifit1^−/− MEFs. Cells were pre-treated with 1 IU/ml of IFNβ for 12 hours or left untreated, and then infected with (A3)VEE/SINV or (G3)VEE/SINV (MOI of 0.1). Supernatants were harvested at indicated times for virus titration ((A3)VEE/SINV: WT + IFNβ versus Ifit1^−/− + IFNβ, 36 and 48 hours post-infection, P < 0.006). Each point represents the average of three independent experiments performed in triplicate, and error bars represent SEM. P values were determined using an unpaired t-test. Dashed lines indicate the limit of detection of the assay. (C and D) Growth kinetics of (G3)VEE/SINV, (A3U24)VEE/SINV, (A3U24;A20U)VEE/SINV, and (A3U24;20_21insC)VEE/SINV viruses in WT (C) and Ifit1^−/− (D) MEFs. Experiments and analysis were performed as in panel A. (E-I) Thermal denaturation of A3, G3, A3U24, A3U24;A20U, and A3U24;20_21insC RNA as measured by CD at 210 nm. RNA was heated from 5 to 95°C at a rate of 1°C/min and readings were collected every 1°C to monitor unfolding.Data is represented as the change in molar ellipticity as a function of temperature (dθ/dT), and red arrows indicate major maxima. One representative experiment of two is shown.

Figure 3. Mutations which alter the secondary structure of the 5′-UTR affect pathogenicity in vivo. (A-D)

Growth kinetics of isogenic TC83 WT and A3G (A and B) or ZPC-738 WT and G3A (C and D) in WT and Ifit1^−/− MEFs. Cells were pre-treated with 10 IU/ml of IFNβ for 12 hours (TC83) or 100 IU/ml of IFNβ for 8 hours (ZPC738), or left untreated, and then infected with respective viruses (MOI of 0.1). (TC83 versus TC83(A3G): WT + IFNβ, 36 and 48 hours post-infection, P < 0.006; ZPC738 vs. ZPC738(G3A): WT + IFNβ, 24 hours post-infection, P < 0.0001). Each point represents the average of two (ZPC-738) or three (TC83) independent experiments performed in triplicate, and error bars represent SEM. P values were determined using the unpaired t-test. Dashed lines indicate the limit of detection of the assay. (E and F) Survival studies of isogenic ZPC-738 WT and G3A (E) and TC83 WT and A3G (F) viruses in WT and Ifit1^−/− mice. Mice were infected s.c. with 10¹ PFU of ZPC-738 (WT, n = 6; Ifit1^−/−, n = 15) or ZPC-738(G3A) (WT, n = 8; Ifit1^−/−, n = 15) and 10⁶ PFU of TC83 (WT, n = 18; Ifit1^−/−, n = 13) or TC83(A3G) (WT, n = 21; Ifit1^−/−, n = 8). ZPC738 versus ZPC738(G3A): WT mice, survival P = 0.0002; mean time to death (MTD) of 5.5 versus 8.3 days, P = 0.0002. ZPC738 versus ZPC738(G3A): Ifit1^−/− mice, MTD of 4.0 versus 5.8 days, P < 0.0001. TC83 versus TC83(A3G): WT mice, survival P < 0.0001; TC83 vs. TC83(A3G): Ifit1^−/− mice, MTD of 8.2 versus 6.3 days, P < 0.003. Experiments were performed twice for ZPC-738 viruses and four times for TC83 viruses. P values for survival were determined as in Fig 1. P values for MTD were determined using an unpaired t-test. (G and H) Growth kinetics of SINV Toto, A5G, and G8U SINV in WT (G) and Ifit1^−/− (H) MEFs. Cells were pre-treated with 1 IU/ml of IFNβ for 12 hours or left untreated, and then infected with respective viruses at an MOI of 0.1. SINV Toto versus A5G: WT MEFs + IFNβ, P < 0.05; SINV Toto versus G8U, WT MEFs + IFNβ, P < 0.05. Experiments and analysis was performed as in panel A.

Figure 4. The nt G3 in the 5′-UTR relieves translational inhibition by altering Ifit1-RNA binding. (A-D)

Luciferase assays of A3 and G3 TRD translation reporters. WT and Ifit1^−/− MEFs were untreated or treated with 100 IU/ml IFNβ for 8 hours, and then electroporated with in vitro synthesized and type 0 capped reporter RNA. Cell lysates were harvested at indicated time points and assayed for luciferase activity. Each bar represents the average of four independent experiments performed in triplicate. WT MEFs + IFNβ: G3 versus A3, P < 0.0004; WT MEFs, no treatment: G3 versus A3, P < 0.005; Ifit1^−/− MEFs + IFNβ, G3 versus A3, P < 0.05 (30, 60, and 120 minutes). Error bars represent the SEM. P values were determined using an unpaired t-test. (E-G) EMSA of A3 and G3 VEEV 5′-UTR RNA bound to recombinant Ifit1. G3 and A3 VEEV 5′-UTR RNA were synthesized in vitro using T7 polymerase (5′-ppp) and then treated with (E) an N-7 methylguanosine capping reagent (Cap 0), (F) an N-7 methylguanosine capping reagent and an exogenous 2′-O methyltranferase (Cap 1), or (G) no enzymes (5′-ppp). All RNA was labeled with biotin and competed with 3 μg of homologous unlabeled RNA. Cap 0 and Cap 1 RNA were heated at 95°C; 5′-ppp RNA were heated at 70°C, as no specific binding was observed after heating at 95°C. Binding assays were performed with 1 μg of Ifit1. EMSA data is representative of at least three independent experiments. Arrows indicate specific binding of RNA to Ifit1 whereas asterisks indicate non-specific binding (not competed with unlabeled RNA). G3 and A3 5′-ppp paired samples were run simultaneously on the same gel and cropped as individual panels for presentation purposes. (H) Quantification of Ifit1-A3/G3 RNA binding by filter-binding assay at 4°C. The fraction bound of A3 Cap 0 (black squares) and G3 Cap 0 (red squares) was normalized to maximum binding and plotted against Ifit1 concentration. Data from A3 (black) and G3 (red) were fitted using the Hill equation. A3 Cap 0 dissociation constant (kD) = 0.030 ± 0.004 μM; G3 Cap 0 kD = 0.091 ± 0.007 μM. One representative experiment of three performed in triplicate is shown.