Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling.

Bats are reservoirs for many RNA viruses that are highly pathogenic in humans yet relatively apathogenic in the natural host. It has been suggested that differences in innate immunity are responsible. The antiviral OAS-RNase L pathway is well characterized in humans, but there is little known about its activation and antiviral activity in bats. During infection, OASs, upon sensing double-stranded RNA (dsRNA), produce 2'-5' oligoadenylates (2-5A), leading to activation of RNase L which degrades viral and host RNA, limiting viral replication. Humans encode three active OASs (OAS1 to -3). Analysis of the Egyptian Rousette bat genome combined with mRNA sequencing from bat RoNi/7 cells revealed three homologous OAS proteins. Interferon alpha treatment or viral infection induced all three OAS mRNAs, but RNase L mRNA is constitutively expressed. Sindbis virus (SINV) or vaccinia virus (VACVΔE3L) infection of wild-type (WT) or OAS1-KO (knockout), OAS2-KO, or MAVS-KO RoNi/7 cells, but not RNase L-KO or OAS3-KO cells, induces robust RNase L activation. SINV replication is 100- to 200-fold higher in the absence of RNase L or OAS3 than in WT cells. However, MAVS-KO had no detectable effect on RNA degradation or replication. Thus, in RoNi/7 bat cells, as in human cells, activation of RNase L during infection and its antiviral activity are dependent primarily on OAS3 while MAVS signaling is not required for the activation of RNase L and restriction of infection. Our findings indicate that OAS proteins serve as pattern recognition receptors (PRRs) to recognize viral dsRNA and that this pathway is a primary response to virus rather than a secondary effect of interferon signaling.IMPORTANCE Many RNA viruses that are highly pathogenic in humans are relatively apathogenic in their bat reservoirs, making it important to compare innate immune responses in bats to those well characterized in humans. One such antiviral response is the OAS-RNase L pathway. OASs, upon sensing dsRNA, produce 2-5A, leading to activation of RNase L which degrades viral and host RNA, limiting viral replication. Analysis of Egyptian Rousette bat sequences revealed three OAS genes expressing OAS1, OAS2, and OAS3 proteins. Interferon treatment or viral infection induces all three bat OAS mRNAs. In these bat cells as in human cells, RNase L activation and its antiviral activity are dependent primarily on OAS3 while MAVS signaling is not required. Importantly, our findings indicate the OAS-RNase L system is a primary response to virus rather than a secondary effect of interferon signaling and therefore can be activated early in infection or while interferon signaling is antagonized.

Li Y ，Dong B ，Wei Z ，Silverman RH ，Weiss SR ... -  《mBio》

Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses.

Li Y ，Banerjee S ，Wang Y ，Goldstein SA ，Dong B ，Gaughan C ，Silverman RH ，Weiss SR ... -  《-》

Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on Basal Oas Gene Expression and Independent of Virus-Induced Interferon.

The oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2',5'-oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2',5'-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2(H126R)) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2(H126R) replicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient (Ifnar1(-/-)) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions of Oas gene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2(H126R) activated RNase L in Ifih1(-/-) BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2(H126R) failed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as in Ifnar1(-/-) BMM, both expressing low basal levels of Oas genes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basal Oas gene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels. The oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basal Oas gene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.

Birdwell LD ，Zalinger ZB ，Li Y ，Wright PW ，Elliott R ，Rose KM ，Silverman RH ，Weiss SR ... -  《-》

The 2'-5'-oligoadenylate synthetase 3 enzyme potently synthesizes the 2'-5'-oligoadenylates required for RNase L activation.

The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS. We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.

Ibsen MS ，Gad HH ，Thavachelvam K ，Boesen T ，Desprès P ，Hartmann R ... -  《-》

Rotavirus Controls Activation of the 2'-5'-Oligoadenylate Synthetase/RNase L Pathway Using at Least Two Distinct Mechanisms.

The innate immune response is the first line of defense of the host cell against a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize, the host innate immune pathways. One of these pathways is the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by double-stranded RNA (dsRNA) to produce 2'-5' oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs, restricting viral infection. It has been recently found that the carboxy-terminal domain (CTD) of rotavirus VP3 has a 2'-5'-phosphodiesterase (PDE) activity that is able to functionally substitute for the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2'-5'-phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated; however, the virus is able to control its activity using at least two distinct mechanisms. A virus-cell interaction that occurs during or before rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Cosilencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles. Rotaviruses represent an important cause of severe gastroenteritis in the young of many animal species, including humans. In this work, we have found that the OAS/RNase L pathway is activated during rotavirus infection, but the virus uses two different strategies to prevent the deleterious effects of this innate immune response of the cell. Early during virus entry, the initial interactions of the viral particle with the cell result in the inhibition of RNase L activity during the first hours of the infection. Later on, once viral proteins are synthesized, the phosphodiesterase activity of VP3 degrades the cellular 2'-5'-oligoadenylates, which are potent activators of RNase L, preventing its activation. This work demonstrates that the OAS/RNase L pathway plays an important role during infection and that the phosphodiesterase activity of VP3 is relevant during the replication cycle of the virus.

Sánchez-Tacuba L ，Rojas M ，Arias CF ，López S ... -  《-》