Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs

Abstract

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

FIG. 1

High virulence of attenuated RVFV strains in IFN-deficient mice. IFNAR^−/− mice lacking the receptor for IFN-α/β (solid triangles) and genotype-matched normal mice (open circles) were infected intraperitoneally with 10⁴ PFU of either wild-type virus ZH548 (A), attenuated strain MP12 (B), or attenuated strain clone 13 (C). Survival was assessed daily for up to 21 days.

FIG. 2

Fulminant hepatitis caused by attenuated clone 13 in IFN-deficient mice. Histology, immunostaining, and in situ hybridization of post mortem liver sections from IFNAR^−/− mice inoculated with 10⁴ PFU of clone 13. (A) Hematoxylin-eosin staining showing perivascular coagulative necrosis and numerous apoptotic nuclei around the portal area. (B) Immunostaining for viral N protein. (C) Loss of glycogen as revealed by periodic acid Schiff staining. (D) In situ hybridization detecting virus-specific nucleic acids in infected (D1) or uninfected (D2) hepatocytes. Magnifications: (A and C) ×360; (B) ×90; (inset) ×225; (D) ×225.

FIG. 3

Attenuated RVFV strains cause viremia in IFN-deficient mice. Growth of virulent virus ZH548 (A), attenuated strain MP12 (B), and attenuated strain clone 13 (C) in IFNAR^−/− mice (solid triangles) and control mice (open circles). Serum samples were collected from six mice per group at the indicated times after infection with 10⁴ PFU and pooled before plaque titration on Vero cells.

FIG. 4

Virulent RVFV strain ZH548 is a poor inducer of IFN-α/β. Aliquots of the pooled serum samples described in Fig. 3 were inactivated at low pH and tested for acid-stable IFN-α/β in a standard bioassay. Sera were from IFNAR^−/− mice (solid triangles) or control mice (open circles) infected with 10⁴ PFU of either wild-type virus ZH548 (A), attenuated strain MP12 (B), or attenuated strain clone 13 (C).

FIG. 5

S segment of RVFV determines IFN inducibility. IFNAR^−/− mice were infected with 10⁴ PFU of either wild-type virus ZH548 (Z/Z/Z), attenuated clone 13 (C/C/C), or the reassortant virus Z/Z/C or C/C/Z. Individual blood samples were collected at the indicated times after infection. Sera from six mice per group were pooled, and aliquots were assayed in parallel for infectious virus in a plaque assay (solid triangles) and for IFN-α/β in a standard bioassay (open circles).