Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs

<div><p>Viral invasion triggers the activation of the host antiviral response. Besides the innate immune response, stress granules (SGs) also act as an additional defense response to combat viral replication. However, many viruses have evolved various strategies to suppress SG formation to facilitate their own replication. Here, we show that viral mRNAs derived from human parainfluenza virus type 3 (HPIV3) infection induce SG formation in an eIF2α phosphorylation- and PKR-dependent manner in which viral mRNAs are sequestered and viral replication is inhibited independent of the interferon signaling pathway. Furthermore, we found that inclusion body (IB) formation by the interaction of the nucleoprotein (N) and phosphoprotein (P) of HPIV3 correlated with SG suppression. In addition, co-expression of P with N_L478A (a point mutant of N, which is unable to form IBs with P) or with NΔN10 (lacking N-terminal 10 amino acids of N, which could form IBs with P but was unable to synthesize or shield viral RNAs) failed to inhibit SG formation, suggesting that inhibition of SG formation also correlates with the capacity of IBs to synthesize and shield viral RNAs. Therefore, we provide a model whereby viral IBs escape the antiviral effect of SGs by concealing their own newly synthesized viral RNAs and offer new insights into the emerging role of IBs in viral replication.</p></div

Proposed mechanism of LacA-mediated MG degradation.

<p>i, pathway I; ii, pathway II.</p

UV-visible spectra of MG before and after treatment with LacA.

<p>UV-visible spectra of MG before and after treatment with LacA.</p

The mass spectra of MG and its degradation products.

<p>(A) MG, <i>m</i>/<i>z</i> 329.20 (retention time 7.23 min). (B) desmethyl MG, <i>m</i>/<i>z</i> 315.18 (retention time 7.01 min). (C) didesmethyl MG, <i>m</i>/<i>z</i> 301.17 (retention time 6.84 min). (D) tridesmethyl MG, <i>m</i>/<i>z</i> 287.16 (retention time 6.65 min). (E) tetradesmethyl MG <i>m</i>/<i>z</i> 273.14 (retention time 6.53 min). (F) (dimethyl amino-phenyl)-phenyl-methanone, <i>m</i>/<i>z</i> 226.12 (retention time 9.14 min). (G) (methyl amino-phenyl)-phenyl-methanone, <i>m</i>/<i>z</i> 212.11 (retention time 8.48 min). (H) (amino phenyl)-phenyl methanone, <i>m</i>/<i>z</i> 198.09 (retention time 7.80 min).</p

The CCD design and the observed responses for MG decolorization.

<p>The CCD design and the observed responses for MG decolorization.</p

ANOVA for the response surface quadratic model.

<p>*Significant (<i>P</i> < 0.05).</p><p>ANOVA for the response surface quadratic model.</p

Relationship between SG formation and innate immune response.

<p>(A) HEK293T cells were transfected with 50 ng IFNβ-Luc reporter and 20 ng TK-Luc reporter for 12 h, then mock-infected or infected with HPIV3 (MOI = 1 or 10) for 24 h. Cells were harvested for a luciferase assay. (B) HEK293T cells were transfected with 50 ng IFNβ-Luc reporter and 20 ng TK-Luc reporter together with the indicated RNA samples for 24 h. Cells were harvested for a luciferase assay. (C) HEK293T cells were transfected with 50 ng IFNβ-Luc reporter and 20 ng TK-Luc reporter together with the indicated plasmid encoding eIF2α and eIF20078-S51A for 24 h, then mock-infected or infected with HPIV3 (MOI = 1) for another 24h. Cells were harvested for a luciferase assay. (D) HEK293T cells with or without G3BP knockdown were transfected with 50 ng IFNβ-Luc reporter and 20 ng TK-Luc reporter for 12 h, then mock-infected or infected with HPIV3 (MOI = 1) for 24 h. Cells were harvested for a luciferase assay. (E) HEK293T cells with or without G3BP knockdown were mock-infected or infected with HPIV3 (MOI = 1) for 24 h. Cells were harvested to extract the nuclei fraction and the cytosol fraction. Protein samples were analyzed via western blot using anti-IRF3, anti-G3BP, anti-HN, anti-GAPDH, and anti-LMNB1 antibodies. Data are represented as means±SD. Student’s t test: * P<0.05, ** P<0.01, *** P<0.001, ns = not significant.</p

LC-TOF MS analysis of MG degraded by LacA.

<p>(A) MG control. (B) MG degradation after 60 min. (C) MG degradation after 180 min.</p

HPIV3 infection induces SG formation.

<p>(A, B, and C) HeLa cells were mock-treated, treated with AS (0.5 mM) for 1 h, or infected with HPIV3 (MOI = 1). At the indicated time points pi, (A) cells were analyzed by confocal microscopy after being immunostained for HPIV3 (purple), TIA-1 (green), and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B) The percentage of cells containing SGs was quantified in three independent experiments. At least 100 cells were counted each time. Data are represented as means ±SD. Student’s t test: * P<0.05, ** P<0.01, *** P<0.001, ns = not significant. (C) Cell lysates were analyzed via western blot using anti-HN, anti-phosphorylated eIF2α, anti-eIF2α, and anti-GAPDH antibodies.</p

CHX treatment induces HPIV3-triggered SG disassembly.

<p>(A and B) HeLa cells were treated with AS (0.5 mM) for 30 min and subsequently treated with or without CHX in the presence of AS for another 1 h. (A) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B) The percentage of cells containing SGs was quantified in three independent experiments. (C-H) HeLa cells were mock-infected or infected with HPIV3 (MOI = 1) for 24 h and subsequently mock-treated or treated with CHX for another 1 h, 2 h, or 3 h. (C) Cells were immnostained for HPIV3 (purple), TIA-1 (green), and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (D) The percentage of cells containing SGs was quantified in three independent experiments. (E) Cell lysates were analyzed via western blot using anti-HN and anti-GAPDH antibodies. (F) Western blots from three independent experiments were quantified and normalized with respect to the amount of GAPDH. (G and H) Total RNA were isolated for qPCR analysis to measure the indicated RNA abundance and normalized to that of GAPDH. Data are represented as means ±SD. Student’s t test: * P<0.05, ** P<0.01, *** P<0.001, ns = not significant.</p