IRF3 Inhibition by Rotavirus NSP1 Is Host Cell and Virus Strain Dependent but Independent of NSP1 Proteasomal Degradation

Abstract

Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.Rotavirus (RV) causes severe dehydrating diarrhea in the young of many mammalian species, and in humans the virus is responsible for approximately 600,000 deaths annually. RV is a member of the Reoviridae family, and virus particles are nonenveloped and icosahedral and contain an 11-segmented double-stranded RNA (dsRNA) genome (13). As a consequence of high error rates of the viral RNA-dependent RNA polymerase and frequent gene reassortment, a large diversity of RV strains constantly circulates in nature. However, despite this diversity RVs are naturally replication restricted in heterologous host species, generally resulting in poor replication (compared to homologous species RV replication) and the inability to cause diarrhea at low dosage in heterologous hosts (8,14,15,23). This natural phenomenon of host range restriction formed the basis for development of several attenuated RV vaccine strains (1, 3, 18). Notably, the mechanistic basis for host range res...

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

“…Although a wealth of knowledge has been gained from mouse studies with nonhuman enteric viruses, these models do not account for evolutionary differences in host and pathogen. Subtle but possibly important differences exist between human and mouse type I and III IFN systems and between human and mouse rotaviruses (18,(35)(36)(37)(38), necessitating functional studies of interactions between human host cells and human enteric pathogens.…”

“…In addition, HRV generally replicates to lower titers than animal rotaviruses in transformed cell lines (2). Therefore, most knowledge of rotavirus-host interactions comes from studies that analyzed animal rotavirus replication in animal models or in transformed cell lines (15)(16)(17)(18). Induction of the adaptive immune system and secretion of IgA are critical factors in protecting the host against rotavirus infection based on in vivo models of animal rotavirus infection (19,20).…”

The Early Interferon Response to Rotavirus Is Regulated by PKR and Depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3

“…In a mouse model of rotavirus infection, RRV replication in gallbladder epithelia correlates with its ability to suppress the IFN response (N. Feng et al, submitted for publication). The rotavirus gene segment encoding the nonstructural protein 1 (NSP1) is an important determinant of host restriction of viral replication both in vitro (17,53) and in vivo (9). Specifically, we along with others have reported a role for NSP1 in regulating the ability of rotavirus to efficiently cause diarrhea in mice (9), spread from animal to animal (9), replicate in vivo and in vitro (4,(15)(16)(17), and antagonize IFN (4,5,17,24,25,53).…”

“…In primary mouse embryonic fibroblasts (MEFs), several heterologous (nonmurine) rotavirus strains, including the bovine UK strain, are replication restricted by the host type I interferon (IFN) response (17,53). In contrast, replication of homologous murine EW virus and the heterologous simian rhesus rotavirus (RRV) strain is insensitive to the presence of the interferon system.…”

Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia

“…A previous study performed by Feng et al (9) demonstrated that both VP4 and NSP1 genes from RRV were required for replication within the mouse biliary tract when comparing RRV and the bovine strain UK. NSP1 has previously been shown to inhibit interferon regulatory factors to suppress type I interferon production (8,22). To date, not all of the rotavirus strains examined in this study have had their NSP1 gene sequenced; thus we cannot rule out the potential involvement of NSP1 in the strains' ability to replicate within biliary cells.…”