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. 2007 May;81(10):4991-9.
doi: 10.1128/JVI.01933-06. Epub 2007 Mar 7.

La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts

Gjon Blakqori  1 , Sophie Delhaye, Matthias Habjan, Carol D Blair, Irma Sánchez-Vargas, Ken E Olson, Ghassem Attarzadeh-Yazdi, Rennos Fragkoudis, Alain Kohl, Ulrich Kalinke, Siegfried Weiss, Thomas Michiels, Peter Staeheli, Friedemann Weber
Affiliations

La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts

Gjon Blakqori et al. J Virol. 2007 May.

Abstract

La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.

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Figures

FIG. 1.
FIG. 1.
Formation of LACV-specific small RNAs during persistent infection of C6/36 mosquito cells. Cells were infected with 10 PFU per cell of LACV and passaged twice weekly, and total RNAs were extracted at given time points. (A) To monitor infection, RNAs were separated by 1.2% agarose gel electrophoresis and hybridized to a negative sense S segment RNA probe. (B) Small RNAs were detected after precipitation of the RNA samples with 5% polyethylene glycol, separation on 16% polyacrylamide gels, and hybridization using the same RNA probe as used in panel A. Arrows show the positions of 20-nucleotide markers which were run on the same gel. p.i., postinfection.
FIG. 2.
FIG. 2.
Time course of persistent LACV infection of mosquito cells. C6/36 cells were infected with 10 PFU per cell of either rLACV or rLACVdelNSs and passaged twice weekly. Viral titers in the supernatants were determined by plaque assay.
FIG. 3.
FIG. 3.
Protection of C6/36 mosquito cells from superinfection. (A) Experimental outline. C6/36 mosquito cells were infected with LACV and maintained for several passages to establish persistent infection (first infection). The persistently infected cells were then superinfected with GFP-expressing reporter viruses (second infection). The reporter viruses were generated by incorporating GFP-RNPs (reconstituted by transfecting BSR-T7/5 cells with expression constructs for N, L, and a GFP minireplicon) into LACV particles as a fourth segment. Both first and second infections involved either wt viruses or delNSs mutants. (B) Results. C6/36 mosquito cells persistently infected with either rLACV or rLACVdelNSs or left uninfected (mock) were superinfected with GFP reporter viruses. At 24 h postinfection, cells were fixed and stained with antibodies against GFP and LACV N protein.
FIG. 4.
FIG. 4.
Effect of LACV NSs on RNAi in insect cells. U4.4 cells were transfected with SFV-Luc SFV-NS1, SFV-TSWV NSs, or SFV-LACV NSs as indicated. At 3 days posttransfection, cells were lysed, and luciferase activities were determined. Mean values and standard deviations from three independent experiments are shown.
FIG. 5.
FIG. 5.
Effect of LACV NSs on IFN induction. (A) Detection of IFN-β mRNA in infected mammalian cells. MEFs isolated from wt or IFNAR0/0 mice were either mock infected or infected with rLACV or rLACVdelNSs. At 18 h postinfection, RNA was extracted and investigated by RT-PCR for the presence of mRNAs for IFN-β, LACV N protein, or β-actin. (B) Suppression of the IFN-β promoter by NSs. Human 293 cells were transfected with luciferase-expressing plasmids under control of either the IFN-β promoter (FF-Luc) or the constitutively active SV40 promoter (REN-Luc), along with the NSs expression plasmid pI.18-HA-LACV-NSs or the empty vector. At 8 h posttransfection, cells were either mock treated or transfected with dsRNA and lysed 18 h later to measure luciferase activity. Mean values and standard deviations from four independent experiments are shown.
FIG. 6.
FIG. 6.
Growth of viruses in IFN-competent and IFN-deficient cells. Primary MEFs derived from wt mice (A), IFNAR0/0 mice (B), or IFN-β0/0 mice (C) were infected with rLACV or rLACVdelNSs at 0.0001 PFU per cell, and virus titers in the supernatants were determined at 24, 48, and 72 h postinfection. Mean values and standard deviations from three independent experiments are shown.
FIG. 7.
FIG. 7.
IFN-dependent virulence of viruses in vivo. Survival of wt (A), IFNAR0/0 (B), or IFN-β0/0 mice (C) infected with 10,000 PFU of rLACV or rLACVdelNSs was monitored. Mice that were moribund or severely paralyzed were killed and scored dead on that day.
FIG. 8.
FIG. 8.
Infection of neurons in vivo. (A) Brain sections of wt mice infected with rLACV (frame 1) or rLACVdelNSs (frames 2 to 4) were stained for viral antigen (green) and the neuron marker NeuN (red). Frames 1 to 3 show sections of the cortex, and frame 3 is a magnification of the section shown in frame 2. Frame 4 shows a section of the thalamus. (B) Brain sections from mice infected with rLACVdelNSs were double stained for viral antigen and markers for astrocytes (GFAP) or macrophages (CD11b).
FIG. 9.
FIG. 9.
Induction of IFN in the mouse brain. ISH with IFN-β or IFN-α probes (see Materials and Methods) was performed on brain sections of mice infected with rLACV or rLACVdelNSs. Macroscopic analysis (A) and microscopic analysis (B) using a combination of ISH to detect IFN-β or IFN-α mRNA (black silver grains) and immunohistochemical analysis for LACV N (brown stain).
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