Showing papers on "NSP1 published in 2009"

Rotavirus NSP1 Inhibits NFκB Activation by Inducing Proteasome-Dependent Degradation of β-TrCP: A Novel Mechanism of IFN Antagonism

Abstract: Mechanisms by which viruses counter innate host defense responses generally involve inhibition of one or more components of the interferon (IFN) system. Multiple steps in the induction and amplification of IFN signaling are targeted for inhibition by viral proteins, and many of the IFN antagonists have direct or indirect effects on activation of latent cytoplasmic transcription factors. Rotavirus nonstructural protein NSP1 blocks transcription of type I IFNα/β by inducing proteasome-dependent degradation of IFN-regulatory factors 3 (IRF3), IRF5, and IRF7. In this study, we show that rotavirus NSP1 also inhibits activation of NFκB and does so by a novel mechanism. Proteasome-mediated degradation of inhibitor of κB (IκBα) is required for NFκB activation. Phosphorylated IκBα is a substrate for polyubiquitination by a multisubunit E3 ubiquitin ligase complex, Skp1/Cul1/F-box, in which the F-box substrate recognition protein is β-transducin repeat containing protein (β-TrCP). The data presented show that phosphorylated IκBα is stable in rotavirus-infected cells because infection induces proteasome-dependent degradation of β-TrCP. NSP1 expressed in isolation in transiently transfected cells is sufficient to induce this effect. Targeted degradation of an F-box protein of an E3 ligase complex with a prominent role in modulation of innate immune signaling and cell proliferation pathways is a unique mechanism of IFN antagonism and defines a second strategy of immune evasion used by rotaviruses.

Rotavirus and reovirus modulation of the interferon response.

Abstract: The mammalian reoviruses and rotaviruses have evolved specific mechanisms to evade the Type I interferon (IFN) antiviral response. Rotavirus likely represses the IFN response by at least 4 mechanisms. First, the rotavirus protein NSP1, most likely functioning as an E3 ligase, can induce proteasome-dependent degradation of the transcription factors IRF3, IRF5, and IRF7 to prevent their induction of IFN. Second, NSP1 can induce proteasome-dependent degradation of the ubiquitin ligase complex protein beta-TrCP, resulting in stabilization of I kappaB and concomitant failure of virus to activate NF-kappaB for induction of IFN. Third, rotavirus may sequester NF-kappaB in viroplasms. And fourth, rotavirus can prevent STAT1 and STAT2 nuclear translocation. The predominant mechanism for rotavirus inhibition of the IFN response is likely both rotavirus strain-specific and cell type-specific. The mammalian reoviruses also display strain-specific differences in their modulation of the IFN response. Reovirus activates RIG-I and IPS-1 for phosphorylation of IRF3. Reovirus-induced activation of MDA5 also participates in induction if IFN-beta, perhaps through activation of NF-kappaB. Reovirus likely inhibits the IFN response by at least 3 virus strain-specific mechanisms. First, the reovirus mu2 protein can induce an unusual nuclear accumulation of IRF9 and repress IFN-stimulated gene (ISG) expression, most likely by disrupting IRF9 function as part of the heterotrimeric transcription factor complex, ISGF3. Second, the reovirus sigma 3 protein can bind dsRNA and prevent activation of the latent antiviral effector protein PKR. And third, genetic approaches have identified the reovirus lambda 2 and sigma 2 proteins in virus strain-specific modulation of the IFN response, but the significance remains unclear. In sum, members of the family Reoviridae have evolved a variety of mechanisms to subvert the host's innate protective response.

Rotavirus Antagonizes Cellular Antiviral Responses by Inhibiting the Nuclear Accumulation of STAT1, STAT2, and NF-κB

Abstract: A vital arm of the innate immune response to viral infection is the induction and subsequent antiviral effects of interferon (IFN). Rotavirus reduces type I IFN induction in infected cells by the degradation of IFN regulatory factors. Here, we show that the monkey rotavirus RRV and human rotavirus Wa also block gene expression induced by type I and II IFNs through a mechanism allowing signal transducer and activator of transcription 1 (STAT1) and STAT2 activation but preventing their nuclear accumulation. In infected cells, this may allow rotavirus to block the antiviral actions of IFN produced early in infection or by activated immune cells. As the intracellular expression of rotavirus nonstructural proteins NSP1, NSP3, and NSP4 individually did not inhibit IFN-stimulated gene expression, their involvement in this process is unlikely. RRV and Wa rotaviruses also prevented the tumor necrosis factor alpha-stimulated nuclear accumulation of NF-κB and NF-κB-driven gene expression. In addition, NF-κB was activated by rotavirus infection, confirming earlier findings by others. As NF-κB is important for the induction of IFN and other cytokines during viral infection, this suggests that rotavirus prevents cellular transcription as a means to evade host responses. To our knowledge, this is the first report of the use of this strategy by a double-stranded RNA virus.

Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses

Abstract: nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs.

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.

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-β) 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-α/β/γ 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-β 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.

Genomic Characterization of Nontypeable Rotaviruses and Detection of a Rare G8 Strain in Delhi, India

Abstract: In the present investigation we molecularly characterized nontypeable rotavirus strains previously identified during surveillance in New Delhi, India. The majority of strains were demonstrated to belong to genotype G1 (54.5%) or P[8] (77.8%) on the basis of nucleotide sequencing of fragments from their VP7 and VP4 genes. The other genotypes detected included G2, G8, G9, G12, and P[4]. A G8P[6] strain, strain DS108, was detected for the first time in northern India. The VP7 gene of DS108 was most homologous with the VP7 gene of a bovine G8 strain, strain A5 (98.9%), indicating its bovine parentage. In contrast, the VP4 gene had a high degree of nucleotide sequence homology (92.9% to 99.1%) with the VP4 genes of human P[6] strains. The VP6 gene and nonstructural genes (NSP1 to NSP3 and NSP5) were most homologous with the VP6 gene and nonstructural genes of human rotaviruses belonging to the DS1 genogroup. Interestingly, the NSP4 gene of DS108 clustered within genotype E6 that until now had only two representative strains, both with G12P[6] specificity (strains RV176-00 and N26-02). Together, these results indicate that G8 strain DS108 belongs to the DS1 genogroup and could be the result of the acquisition of the VP7, VP4, and NSP4 genes by a human G2P[4] strain from more than one donor, similar to the evolution of G12P[6] strain RV176-00. The present study highlights the importance of characterizing multiple genes of nontypeable rotavirus strains to detect novel strains and get a more complete picture of rotavirus evolution.

Rotavirus Antagonism of the Innate Immune Response

Abstract: Rotavirus is a primary cause of severe dehydrating gastroenteritis in infants and young children. The virus is sensitive to the antiviral effects triggered by the interferon (IFN)-signaling pathway, an important component of the host cell innate immune response. To counteract these effects, rotavirus encodes a nonstructural protein (NSP1) that induces the degradation of proteins involved in regulating IFN expression, such as members of the IFN regulatory factor (IRF) family. In some instances, NSP1 also subverts IFN expression by causing the degradation of a component of the E3 ubiquitin ligase complex responsible for activating NF-κB. By antagonizing multiple components of the IFN-induction pathway, NSP1 aids viral spread and contributes to rotavirus pathogenesis.

Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus

Abstract: An infectious clone (pBR-XJ160) was constructed using the full-length cDNA of the Sindbis-like XJ-160 virus. Two nucleotide mutations, causing amino acid changes at residue 169 from Lys to Arg and at residue 173 from Thr to Ile in the nonstructural protein (nsP) 1 coding region, strongly influenced the infectivity of in vitro-synthesized RNA. We used site-directed mutagenesis to obtain clones encoding a change to Arg at residue 169 of nsP1 (pBR-169), a change to Ile at residue 173 (pBR-173), or both changes (pBR-6973). Infectivity of RNA from pBR-169 was abolished, but viral forms BR-173 and BR-6973 were obtained from pBR-173 and pBR-6973, respectively. Further, BR-173 exhibited higher propagation than BR-XJ160 in cell culture and higher neurovirulence in a suckling mouse model. BR-6973 possessed an intermediate phenotype. BR-173 and BR-6973 showed increased sensitivity to 3-deazaadenosine (3-DZA), which inhibits S-adenosylhomocysteine hydrolase. Thus, mutagenesis at residue 169 in the nsP1 region of XJ-160 is lethal, but mutation at residue 173 from Thr to Ile enhances viral infectivity and neurovirulence and suppresses the lethal effect of the mutation at residue 169. These mutations might be associated with the RNA methyltransferase (MTase) activity of nsP1.

Innate immune responses elicited by Reovirus and Rotavirus.

Abstract: The mammalian orthoreoviruses (called simply reoviruses) are the type species of the Orthoreovirus genus, which also contains viruses that infect birds and reptiles. The reoviruses and rotaviruses are the subject of this chapter. The effect of NSP1 can be linked to its role as an antagonist of interferon (IFN) expression. Application of global approaches for identification of reovirus-induced IFN-stimulated genes (ISGs) should provide additional ISG candidates responsible for IFN’s effects on viral replication and possibly apoptosis induction. Recent reports point to NSP1 as the sole rotavirus gene product responsible for countering IFN-dependent innate immune responses. Rotavirus NSP1 is an antagonist of the IFN signaling pathway, most likely functioning through an E3 Ub ligase activity that induces the ubiquitination and proteasomal proteolysis of several members of the IRF family. Many viruses encode proteins that interfere with Jak-Stat signaling. However, there is no published evidence for similar disruption by reoviruses. Like analysis of strain-specific differences in IFN-β induction, genetic analyses using reassortant reoviruses identified the M1, L2, and S2 genes as determinants of strain-specific differences in sensitivity to the antiviral effects of IFN-β in cardiac myocytes.

Rotavirus gene recombined strain and construction method and application thereof

Abstract: The invention discloses a rotavirus gene recombined strain and a construction method and application thereof, belonging to the field of rotavirus diagnosis and control. The rotavirus gene recombined strain uses VP1, VP2, VP3, VP4, NSP1, VP6, NSP2, NSP3, NSP4 and NSP5 genes of an ovine rotavirus lentogen strain as the gene frame of the recombined strain and is recombined with VP7 gene segments of a bovine rotavirus strain, and the microbial preservation number is CGMCC No.3102. The combined application of recombined strains R191 and LLR-85 can constitute a divalent attenuated live vaccine containing main serological types G6 and G10 of the bovine rotavirus. The immunogenicity and the virus liberation of two vaccine candidate strains in a newborn calf in-vivo are evaluated, and the result shows that the vaccine candidate strains in the newborn calf in-vivo has good immunogenicity and very low virus liberation rate.