Variation in antagonism of the interferon response to rotavirus NSP1 results in differential infectivity in mouse embryonic fibroblasts

Abstract

Rotavirus NSP1 has been shown to function as an E3 ubiquitin ligase that mediates proteasome-dependent degradation of interferon (IFN) regulatory factors (IRF), including IRF3, -5, and -7, and suppresses the cellular type I IFN response. However, the effect of rotavirus NSP1 on viral replication is not well defined. Prior studies used genetic analysis of selected reassortants to link NSP1 with host range restriction in the mouse, suggesting that homologous and heterologous rotaviruses might use their different abilities to antagonize the IFN response as the basis of their host tropisms. Using a mouse embryonic fibroblast (MEF) model, we demonstrate that heterologous bovine (UK and NCDV) and porcine (OSU) rotaviruses fail to effectively degrade cellular IRF3, resulting in IRF3 activation and beta IFN (IFN-beta) secretion. As a consequence of this failure, replication of these viruses is severely restricted in IFN-competent wild-type, but not in IFN-deficient (IFN-alpha/beta/gamma receptor- or STAT1-deficient) MEFs. On the other hand, homologous murine rotaviruses (ETD or EHP) or the heterologous simian rotavirus (rhesus rotavirus [RRV]) efficiently degrade cellular IRF3, diminish IRF3 activation and IFN-beta secretion and are not replication restricted in wild-type MEFs. Genetic reassortant analysis between UK and RRV maps the distinctive phenotypes of IFN antagonism and growth restriction in wild-type MEFs to NSP1. Therefore, there is a direct relationship between the replication efficiencies of different rotavirus strains in MEFs and strain-related variations in NSP1-mediated antagonism of the type I IFN response.

FIG. 1.

Virus yield in wild-type or IFN-deficient MEFs following infection with the indicated strains of rotavirus. Wild-type 129SV, IFNR KO, or STAT1 KO MEFs were infected with different rotavirus strains at an MOI of 1 for 24 h. The virus yield after infection was determined by focus-forming assay in MA104 cells and expressed as FFU/ml (see Materials and Methods). Each bar represents the average from quadruple data points plus the standard deviation. *, significant difference in the virus yield compared to IFNR KO and STAT1 KO MEFs (P < 0.01) by ANOVA and Scheffe test.

FIG. 2.

IFN-β secretion from wild-type MEFs following infection with different strains of rotavirus. Wild-type MEFs were infected with UK, RRV, ETD, UK-like reassortant 4-1-1, and RRV-like reassortant 24-1-1 at an MOI of 10 for 16 h. IFN-β secretion in the SN was determined by ELISA. Each bar represents quadruple data points plus the standard deviation. *, statistically significant difference between groups by ANOVA and Scheffe test.

FIG. 3.

IRF3 nuclear translocation in wild-type MEFs following infection with the indicated strains of rotavirus. Wild-type MEFs were infected with the UK (A and B), RRV (C and D), ETD (E and F), UK-like reassortant 4-1-1 (G and H), or RRV-like reassortant 24-1-1 (I and J) strain, and IFNR KO MEFs were infected with UK (K and L) at an MOI of 0.5 for 6 h. The cells were stained for IRF3 (green), rotavirus VP6 (red), and nuclei (blue). The panels on the left show all three colors, except panel K, which shows only red and green, and the panels on the right show only IRF3 (green). The small arrows indicate antigen-positive cells with or without IRF3 nuclear translocation. The large arrows indicate antigen-negative cells with IRF3 nuclear translocation. Magnification, ×600.

FIG. 4.

Immune blot analysis of total IRF3 and pS396 IRF3 in wild-type MEFs infected with the indicated strains of rotavirus. Wild-type MEFs were infected with selected rotaviruses at an MOI of 30 for 16 h or mock infected. Proteins in the cell lysates were analyzed for total IRF3, pS396 IRF3 (pIRF3), and β-actin by immune blotting.

FIG. 5.

Role of viral replication in UK virus-mediated activation of the IFN response. NIH 3T3 cells were infected with equal amounts of live (UK) or psoralen-inactivated (i-UK) virus as indicated, and total protein lysates were examined by immunoblotting at 6 h p.i.