Febrile temperature activates the innate immune response by promoting aberrant influenza A virus RNA synthesis

Abstract

Fever during influenza A virus (IAV) infection is triggered by the innate immune response. Various factors contribute to this response, including IAV mini viral RNAs (mvRNA), which trigger RIG-I signaling when their replication and transcription are dysregulated by template loops (t-loop). It is presently not well understood whether the fever response to IAV infection impacts subsequent viral replication and innate immune activation. Here we show that IAV infection at temperatures that simulate fever leads to increased antiviral signaling in H1N1 and H3N2 infections. Mathematical modeling and experimental analyses reveal that differential IAV nucleoprotein and RNA polymerase production increase mvRNA and interferon production. Moreover, at the higher infection temperature, mvRNAs with dysregulating t-loops contribute most to the innate immune activation. We propose that fever during IAV infection can establish a positive feedback loop in which elevated aberrant RNA synthesis and innate immune activation can contribute to the dysregulation of cytokine production.

Keywords: RIG-I; RNA polymerase; capped cRNA; fever; influenza A virus; interferon; mini viral RNA; mvRNA; temperature; template loop.

Fig. 1:. The single infection cycle model of influenza A Virus (IAV) reveals impact of NP dynamics on innate immune activation.

a, Schematic of IAV replication and transcription during IAV infection. The viral ribonucleoprotein (vRNP) consists of a copy of the viral RNA-dependent RNA polymerase (RdRp), vRNA (orange), and NP (purple). The RdRp consists of three subunits: PB1 (blue), PA (green) and PB2 (pink). Canonical transcription of vRNA molecules produces mRNAs, while replication of vRNA produces cRNAs (purple). cRNA molecules are encapsidated into cRNPs. Non-canonical transcription of vRNAs can produce ccRNAs, while non-canonical replication of vRNA molecules produce mcRNAs. Also, cRNA molecules can be replicated into mvRNAs. The mcRNAs and mvRNAs can be transcribed and replicated (dotted circles). b, Schematic illustrating the processes and variables (see Table S2) incorporated into the model. c, Simulation of IAV infection cycle and prediction of NP and polymerase protein levels per cell. d, Simulation of IAV infection cycle and prediction of mvRNA levels per cell. e, Replication rate of aberrant and full-length products driven by the NP/polymerase ratio. f, Heatmap on left shows RIG-I activity as function of NP and polymerase gene transcription. Plot on right shows RIG-I activity as function of the ratio between the NP and polymerase proteins that follows from the simulations in left plot. Arrow indicates shift in RIG-I activation after fever. g, Analysis of IFNB promoter activity and vRNA levels using primer extension during the replication of a segment 5-based 246-nt RNA template by the WSN polymerases. NP mvRNA levels were analyzed by RT-PCR. NP expression was assessed by western blot (bottom gel). n = 3 biologically independent experiments.

Fig. 2:. Elevated temperature affects both innate immune response and aberrant RNA synthesis during infection.

a, Schematic of experimental design used for viral infection of A549 cells in cells adapted at 37°C and 39°C. b, Volcano plot illustrating differentially expressed genes in A549 cells infected with A/WSN/33 virus at an MOI of 3. RNA-seq was performed on 3 biological replicates. c, Reactome pathway enrichment analysis of the differentially expressed genes. d, Growth kinetics of lab adapted WSN virus in A549 cells at different temperatures (37°C, or 39°C). A549 cells were infected at an MOI of 3 at 37°C and 39°C. The supernatants of the infected cells were harvested at the indicated times, and the virus titers were determined by performing plaque assays in MDCK cells at 37°C. e,f, Viral protein expression analyzed using western blot, the bar graph depicts the quantified relative expression level. g, Schematic of RNA detection using Cas13. h, Detection of NP-61 mvRNA using Cas13 assay in fractionated A549 cells infected with WSN. Graph shows the copy number of mvRNA NP-61 in infected cells at 37°C and 39°C. Data are shown as a mean of 3 independent experiments. Error bars indicate standard deviation. The P values were determined by using an unpaired t test. (*P < 0.05).

Fig. 3:. Differential NP and polymerase protein expression at elevated temperature during infection.

A549 cells adapted at 37°C and 39°C were infected with an MOI of 1 of A/WSN/33 virus and RNA samples were taken at different time points post-infection. c,d, Normalization of vRNA levels relative to mRNA levels for the NP and PA full length segments. e,f, Cell lysates were collected at different time points and analyzed by immunoblotting. Lower panel show the quantified western blot data. g,h, Detection of ccRNA and cRNA by TSO-based RT-PCR. Quantification of fold-change difference in ccRNA and cRNA levels produced at 39°C versus 37°C. Data are shown as the mean of 3 independent experiments. Error bars indicate the standard deviation. The P values were determined by using an unpaired t test.

Fig.4:. mvRNAs induce increased innate immune signaling at higher temperature.

a,b,c, Analysis of IFN-β promoter activity induced by the replication of segment 5, 3 or 2 mvRNAs 24 hours post-transfection of BM18 IAV RNA polymerase. PB1, NP and tubulin expression was analyzed by western blot. d, Normalized vRNA levels for BM18 polymerase relative to WSN polymerase. e, Normalized mRNA levels for BM18 polymerase relative to WSN polymerase. f, ATPase activity of recombinant RIG-I was assessed in the presence of in vitro transcribed PA66 mvRNA, dephosphorylated PA66 mvRNA, or a no RNA control. Data are shown as the mean of 3 independent experiments. Error bars indicate standard deviation. P values were determined using a two-sided, unpaired t-test.

Fig. 5:. Temperature-driven modulation of innate immunity aligns with responses to other IAV strains.

a, Analysis of IFN-β promoter activity, 12 hpi, in HEK-luc cell line adapted at 37°C and 39°C, infected with WSN, Melbourne46, Denver57, Cal09, HK68, and Brisbane07 IAV strains at an MOI of 3. The NP and tubulin expression was analyzed by western blot. b, IFN-β promoter activity was assessed in HEK-luc cells 12 hpi. The cells were adapted to 37°C and 39°C and infected at an MOI of 1 with either the WSN strain or the T677A mutant virus strain. The NP and tubulin expression was analyzed by western blot. Data are shown as the mean of 6 independent experiments. Error bars indicate standard deviation. P values were determined using a two-sided, unpaired t-test. c, An increase in infection temperature can differently impact polymerase and NP expression, and this imbalance may contribute to the synthesis of mvRNAs. d, Formation of mvRNAs is influenced by both temperature and the viral strain infecting the host, which can impact activation of the innate immune response, and cytokine and chemokine expression. A fever start may create a positive feedback loop that leads to increased mvRNA production and innate immune activation, and which may ultimately contribute to a cytokine storm. The likelihood for a cytokine storm may be modulated by risk and other host and viral factors.