Identification of poly(ADP-ribose) polymerase 9 (PARP9) as a noncanonical sensor for RNA virus in dendritic cells.

Innate immune cells are critical in protective immunity against viral infections, involved in sensing foreign viral nucleic acids. Here we report that the poly(ADP-ribose) polymerase 9 (PARP9), a member of PARP family, serves as a non-canonical sensor for RNA virus to initiate and amplify type I interferon (IFN) production. We find knockdown or deletion of PARP9 in human or mouse dendritic cells and macrophages inhibits type I IFN production in response to double strand RNA stimulation or RNA virus infection. Furthermore, mice deficient for PARP9 show enhanced susceptibility to infections with RNA viruses because of the impaired type I IFN production. Mechanistically, we show that PARP9 recognizes and binds viral RNA, with resultant recruitment and activation of the phosphoinositide 3-kinase (PI3K) and AKT3 pathway, independent of mitochondrial antiviral-signaling (MAVS). PI3K/AKT3 then activates the IRF3 and IRF7 by phosphorylating IRF3 at Ser385 and IRF7 at Ser437/438 mediating type I IFN production. Together, we reveal a critical role for PARP9 as a non-canonical RNA sensor that depends on the PI3K/AKT3 pathway to produce type I IFN. These findings may have important clinical implications in controlling viral infections and viral-induced diseases by targeting PARP9.

Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis.

Being opportunistic intracellular pathogens, viruses are dependent on the host for their replication. They hijack host cellular machinery for their replication and survival by targeting crucial cellular physiological pathways, including transcription, translation, immune pathways, and apoptosis. Immediately after translation, the host and viral proteins undergo a process called post-translational modification (PTM). PTMs of proteins involves the attachment of small proteins, carbohydrates/lipids, or chemical groups to the proteins and are crucial for the proteins' functioning. During viral infection, host proteins utilize PTMs to control the virus replication, using strategies like activating immune response pathways, inhibiting viral protein synthesis, and ultimately eliminating the virus from the host. PTM of viral proteins increases solubility, enhances antigenicity and virulence properties. However, RNA viruses are devoid of enzymes capable of introducing PTMs to their proteins. Hence, they utilize the host PTM machinery to promote their survival. Proteins from viruses belonging to the family: Togaviridae, Flaviviridae, Retroviridae, and Coronaviridae such as chikungunya, dengue, zika, HIV, and coronavirus are a few that are well-known to be modified. This review discusses various host and virus-mediated PTMs that play a role in the outcome during the infection.

Incoming RNA virus nucleocapsids containing a 5'-triphosphorylated genome activate RIG-I and antiviral signaling.

Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases: RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded RNAs (dsRNAs) with a triphosphorylated 5' (5'ppp) terminus. However, the identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5'ppp dsRNA "panhandle" structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and superresolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection and establish viral nucleocapsids with a 5'ppp dsRNA panhandle as a RIG-I activator.

Paradoxical interplay of viral and cellular functions.

Some cellular editing functions can restrict the replication of some viruses but contribute to completion of the life cycle of others. A recent study has identified an isoform of the adenosine deaminase acting on RNA type 1 (ADAR 1) as required for embryogenesis, and as a restriction factor for a number of important RNA virus pathogens. The dual implication of key cellular functions in the innate immunity against viruses, or, paradoxically, as mediators of virus replication is interpreted in the light of the concept of virus-host coevolution and tinkering proposed for general evolution by François Jacob decades ago.

Tipping the balance: antagonism of PKR kinase and ADAR1 deaminase functions by virus gene products.

The protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA (ADAR1) are interferon-inducible enzymes that play important roles in biologic processes including the antiviral actions of interferons, signal transduction, and apoptosis. PKR catalyzes the RNA-dependent phosphorylation of protein synthesis initiation factor eIF-2 alpha, thereby leading to altered translational patterns in interferon-treated and virus-infected cells. PKR also modulates signal transduction responses, including the induction of interferon. ADAR1 catalyzes the deamination of adenosine (A) to generate inosine (I) in RNAs with double-stranded character. Because I is recognized as G instead of A, A-to-I editing by ADAR1 can lead to genetic recoding and altered RNA structures. The importance of PKR and ADAR1 in innate antiviral immunity is illustrated by a number of viruses that encode either RNA or protein viral gene products that antagonize PKR and ADAR1 enzymatic activity, localization, or stability.

B-cell-activating factor expressions in salivary epithelial cells after dsRNA virus infection depends on RNA-activated protein kinase activation.

B-cell-activating factor (BAFF) plays a key role in promoting activation of autoimmune B cells. This cytokine may be expressed in and secreted by salivary gland epithelial cells (SGEC) after stimulation with type I IFN or viral or synthetic dsRNA. Because this BAFF expression depends only in part on endosomal TLR and type I IFN, we investigated whether other dsRNA sensors could be implicated in BAFF expression. Using human SGEC, we confirmed the partial dependence of BAFF expression on TLR-3 by replicating the partial inhibition of BAFF expression observed upon endosomal inhibition using TLR-3 or Toll/IL-1R domain-containing protein inducing IFN-beta silencing mRNA, but not with TLR-7 silencing mRNA. Melanoma differentiation-associated gene 5 silencing mRNA had no effect on BAFF expression, but retinoic acid-inducible gene I silencing mRNA had a slight effect observed following infection with dsRNA reovirus-1. Inhibition of RNA-activated protein kinase (PKR) by 2-aminopurine completely abolished both BAFF mRNA and protein production after reovirus-1 infection and poly(I:C) stimulation through NF-kappaB and p38 MAPK pathways, with the latter implicated only after poly(I:C) stimulation. Thus, PKR is the dsRNA sensor implicated in BAFF induction in SGEC after dsRNA stimulation. In autoimmune diseases, PKR may be an interesting target for preventing BAFF following the induction of innate immunity.

RNA viruses and the mitogenic Raf/MEK/ERK signal transduction cascade.

The Raf/MEK/ERK signal transduction cascade belongs to the mitogen-activated protein kinase (MAPK) cascades. Raf/MEK/ERK signaling leads to stimulus-specific changes in gene expression, alterations in cell metabolism or induction of programmed cell death (apoptosis), and thus controls cell differentiation and proliferation. It is induced by extracellular agents, including pathogens such as RNA viruses. Many DNA viruses are known to induce cellular signaling via this pathway. As these pathogens partly use the DNA synthesis machinery for their replication, they aim to drive cells into a proliferative state. In contrast, the consequences of RNA virus-induced Raf/MEK/ERK signaling were less clear for a long time, but since the turn of the century the number of publications on this topic has rapidly increased. Research on this virus/host-interaction will broaden our understanding of its relevance in viral replication. This important control center of cellular responses is differently employed to support the replication of several important human pathogenic RNA viruses including influenza, Ebola, hepatitis C and SARS corona viruses.

Cell type-specific involvement of RIG-I in antiviral response.

Toll-like receptors (TLRs) play an important role in antiviral response by recognizing viral components. Recently, a RNA helicase, RIG-I, was also suggested to recognize viral double-stranded RNA. However, how these molecules contribute to viral recognition in vivo is poorly understood. We show by gene targeting that RIG-I is essential for induction of type I interferons (IFNs) after infection with RNA viruses in fibroblasts and conventional dendritic cells (DCs). RIG-I induces type I IFNs by activating IRF3 via IkappaB kinase-related kinases. In contrast, plasmacytoid DCs, which produce large amounts of IFN-alpha, use the TLR system rather than RIG-I for viral detection. Taken together, RIG-I and the TLR system exert antiviral responses in a cell type-specific manner.