Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor.

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Protein-Ligand Interactions in Scarcity: The Stringent Response from Bacteria to Metazoa, and the Unanswered Questions.

The stringent response, originally identified in Escherichia coli as a signal that leads to reprogramming of gene expression under starvation or nutrient deprivation, is now recognized as ubiquitous in all bacteria, and also as part of a broader survival strategy in diverse, other stress conditions. Much of our insight into this phenomenon derives from the role of hyperphosphorylated guanosine derivatives (pppGpp, ppGpp, pGpp; guanosine penta-, tetra- and tri-phosphate, respectively) that are synthesized on starvation cues and act as messengers or alarmones. These molecules, collectively referred to here as (p)ppGpp, orchestrate a complex network of biochemical steps that eventually lead to the repression of stable RNA synthesis, growth, and cell division, while promoting amino acid biosynthesis, survival, persistence, and virulence. In this analytical review, we summarize the mechanism of the major signaling pathways in the stringent response, consisting of the synthesis of the (p)ppGpp, their interaction with RNA polymerase, and diverse factors of macromolecular biosynthesis, leading to differential inhibition and activation of specific promoters. We also briefly touch upon the recently reported stringent-like response in a few eukaryotes, which is a very disparate mechanism involving MESH1 (Metazoan SpoT Homolog 1), a cytosolic NADPH phosphatase. Lastly, using ppGpp as an example, we speculate on possible pathways of simultaneous evolution of alarmones and their multiple targets.

Suppression of Innate Immunity by the Hepatitis C Virus (HCV): Revisiting the Specificity of Host-Virus Interactive Pathways.

The hepatitis C virus (HCV) is a major causative agent of hepatitis that may also lead to liver cancer and lymphomas. Chronic hepatitis C affects an estimated 2.4 million people in the USA alone. As the sole member of the genus Hepacivirus within the Flaviviridae family, HCV encodes a single-stranded positive-sense RNA genome that is translated into a single large polypeptide, which is then proteolytically processed to yield the individual viral proteins, all of which are necessary for optimal viral infection. However, cellular innate immunity, such as type-I interferon (IFN), promptly thwarts the replication of viruses and other pathogens, which forms the basis of the use of conjugated IFN-alpha in chronic hepatitis C management. As a countermeasure, HCV suppresses this form of immunity by enlisting diverse gene products, such as HCV protease(s), whose primary role is to process the large viral polyprotein into individual proteins of specific function. The exact number of HCV immune suppressors and the specificity and molecular mechanism of their action have remained unclear. Nonetheless, the evasion of host immunity promotes HCV pathogenesis, chronic infection, and carcinogenesis. Here, the known and putative HCV-encoded suppressors of innate immunity have been reviewed and analyzed, with a predominant emphasis on the molecular mechanisms. Clinically, the knowledge should aid in rational interventions and the management of HCV infection, particularly in chronic hepatitis.

Special Issue: Structure, Function and Evolution of Protein Domains.

Essentially, all proteins perform their biological roles through the use of specific domains that number in the hundreds, if not thousands [...].

Mechanisms of Viral Degradation of Cellular Signal Transducer and Activator of Transcription 2.

Virus infection of eukaryotes triggers cellular innate immune response, a major arm of which is the type I interferon (IFN) family of cytokines. Binding of IFN to cell surface receptors triggers a signaling cascade in which the signal transducer and activator of transcription 2 (STAT2) plays a key role, ultimately leading to an antiviral state of the cell. In retaliation, many viruses counteract the immune response, often by the destruction and/or inactivation of STAT2, promoted by specific viral proteins that do not possess protease activities of their own. This review offers a summary of viral mechanisms of STAT2 subversion with emphasis on degradation. Some viruses also destroy STAT1, another major member of the STAT family, but most viruses are selective in targeting either STAT2 or STAT1. Interestingly, degradation of STAT2 by a few viruses requires the presence of both STAT proteins. Available evidence suggests a mechanism in which multiple sites and domains of STAT2 are required for engagement and degradation by a multi-subunit degradative complex, comprising viral and cellular proteins, including the ubiquitin-proteasomal system. However, the exact molecular nature of this complex and the alternative degradation mechanisms remain largely unknown, as critically presented here with prospective directions of future study.

Inhibition of Viral RNA-Dependent RNA Polymerases by Nucleoside Inhibitors: An Illustration of the Unity and Diversity of Mechanisms.

RNA-dependent RNA polymerase (RdRP) is essential for the replication and expression of RNA viral genomes. This class of viruses comprise a large number of highly pathogenic agents that infect essentially all species of plants and animals including humans. Infections often lead to epidemics and pandemics that have remained largely out of control due to the lack of specific and reliable preventive and therapeutic regimens. This unmet medical need has led to the exploration of new antiviral targets, of which RdRP is a major one, due to the fact of its obligatory need in virus growth. Recent studies have demonstrated the ability of several synthetic nucleoside analogs to serve as mimics of the corresponding natural nucleosides. These mimics cause stalling/termination of RdRP, or misincorporation, preventing virus replication or promoting large-scale lethal mutations. Several such analogs have received clinical approval and are being routinely used in therapy. In parallel, the molecular structural basis of their inhibitory interactions with RdRP is being elucidated, revealing both traditional and novel mechanisms including a delayed chain termination effect. This review offers a molecular commentary on these mechanisms along with their clinical implications based on analyses of recent results, which should facilitate the rational design of structure-based antiviral drugs.

An Analytical Review of the Structural Features of Pentatricopeptide Repeats: Strategic Amino Acids, Repeat Arrangements and Superhelical Architecture.

Tricopeptide repeats are common in natural proteins, and are exemplified by 34- and 35-residue repeats, known respectively as tetratricopeptide repeats (TPRs) and pentatricopeptide repeats (PPRs). In both classes, each repeat unit forms an antiparallel bihelical structure, so that multiple such units in a polypeptide are arranged in a parallel fashion. The primary structures of the motifs are nonidentical, but amino acids of similar properties occur in strategic positions. The focus of the present work was on PPR, but TPR, its better-studied cousin, is often included for comparison. The analyses revealed that critical amino acids, namely Gly, Pro, Ala and Trp, were placed at distinct locations in the higher order structure of PPR domains. While most TPRs occur in repeats of three, the PPRs exhibited a much greater diversity in repeat numbers, from 1 to 30 or more, separated by spacers of various sequences and lengths. Studies of PPR strings in proteins showed that the majority of PPR units are single, and that the longer tandems (i.e., without space in between) occurred in decreasing order. The multi-PPR domains also formed superhelical vortices, likely governed by interhelical angles rather than the spacers. These findings should be useful in designing and understanding the PPR domains.

Evolution of Protein Structure and Stability in Global Warming.

This review focuses on the molecular signatures of protein structures in relation to evolution and survival in global warming. It is based on the premise that the power of evolutionary selection may lead to thermotolerant organisms that will repopulate the planet and continue life in general, but perhaps with different kinds of flora and fauna. Our focus is on molecular mechanisms, whereby known examples of thermoresistance and their physicochemical characteristics were noted. A comparison of interactions of diverse residues in proteins from thermophilic and mesophilic organisms, as well as reverse genetic studies, revealed a set of imprecise molecular signatures that pointed to major roles of hydrophobicity, solvent accessibility, disulfide bonds, hydrogen bonds, ionic and π-electron interactions, and an overall condensed packing of the higher-order structure, especially in the hydrophobic regions. Regardless of mutations, specialized protein chaperones may play a cardinal role. In evolutionary terms, thermoresistance to global warming will likely occur in stepwise mutational changes, conforming to the molecular signatures, such that each "intermediate" fits a temporary niche through punctuated equilibrium, while maintaining protein functionality. Finally, the population response of different species to global warming may vary substantially, and, as such, some may evolve while others will undergo catastrophic mass extinction.

The Uniqueness of Tryptophan in Biology: Properties, Metabolism, Interactions and Localization in Proteins.

Tryptophan (Trp) holds a unique place in biology for a multitude of reasons. It is the largest of all twenty amino acids in the translational toolbox. Its side chain is indole, which is aromatic with a binuclear ring structure, whereas those of Phe, Tyr, and His are single-ring aromatics. In part due to these elaborate structural features, the biosynthetic pathway of Trp is the most complex and the most energy-consuming among all amino acids. Essential in the animal diet, Trp is also the least abundant amino acid in the cell, and one of the rarest in the proteome. In most eukaryotes, Trp is the only amino acid besides Met, which is coded for by a single codon, namely UGG. Due to the large and hydrophobic π-electron surface area, its aromatic side chain interacts with multiple other side chains in the protein, befitting its strategic locations in the protein structure. Finally, several Trp derivatives, namely tryptophylquinone, oxitriptan, serotonin, melatonin, and tryptophol, have specialized functions. Overall, Trp is a scarce and precious amino acid in the cell, such that nature uses it parsimoniously, for multiple but selective functions. Here, the various aspects of the uniqueness of Trp are presented in molecular terms.

Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda N-cI- Prophages.

The bacterial virus lambda (λ) is a temperate bacteriophage that can lysogenize host Escherichia coli (E. coli) cells. Lysogeny requires λ repressor, the cI gene product, which shuts off transcription of the phage genome. The λ N protein, in contrast, is a transcriptional antiterminator, required for expression of the terminator-distal genes, and thus, λ N mutants are growth-defective. When E. coli is infected with a λ double mutant that is defective in both N and cI (i.e., λN-cI-), at high multiplicities of 50 or more, it forms polylysogens that contain 20-30 copies of the λN-cI- genome integrated in the E. coli chromosome. Early studies revealed that the polylysogens underwent "conversion" to long filamentous cells that form tiny colonies on agar. Here, we report a large set of altered biochemical properties associated with this conversion, documenting an overall degeneration of the bacterial envelope. These properties reverted back to those of nonlysogenic E. coli as the metastable polylysogen spontaneously lost the λN-cI- genomes, suggesting that conversion is a direct result of the multiple copies of the prophage. Preliminary attempts to identify lambda genes that may be responsible for conversion ruled out several candidates, implicating a potentially novel lambda function that awaits further studies.

Retraction Note: Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling.

The Editor has retracted this article [1] following an investigation by the University of South Alabama which found.

Molecular Interactions between Pathogens and the Circadian Clock.

The daily periodicity of the Earth's rotation around the Sun, referred to as circadian (Latin "circa" = about, and "diem" = day), is also mirrored in the behavior and metabolism of living beings. The discovery that dedicated cellular genes control various aspects of this periodicity has led to studies of the molecular mechanism of the circadian response at the cellular level. It is now established that the circadian genes impact on a large network of hormonal, metabolic, and immunological pathways, affecting multiple aspects of biology. Recent studies have extended the role of the circadian system to the regulation of infection, host-pathogen interaction, and the resultant disease outcome. This critical review summarizes our current knowledge of circadian-pathogen interaction at both systemic and cellular levels, but with emphasis on the molecular aspects of the regulation. Wherever applicable, the potential of a direct interaction between circadian factors and pathogenic macromolecules is also explored. Finally, this review offers new directions and guidelines for future research in this area, which should facilitate progress.

Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-ß. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. IMPORTANCE: The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.

Induction of interferon-stimulated genes by IRF3 promotes replication of Toxoplasma gondii.

Innate immunity is the first line of defense against microbial insult. The transcription factor, IRF3, is needed by mammalian cells to mount innate immune responses against many microbes, especially viruses. IRF3 remains inactive in the cytoplasm of uninfected cells; upon virus infection, it gets phosphorylated and then translocates to the nucleus, where it binds to the promoters of antiviral genes and induces their expression. Such genes include type I interferons (IFNs) as well as Interferon Stimulated Genes (ISGs). IRF3-/- cells support enhanced replication of many viruses and therefore, the corresponding mice are highly susceptible to viral pathogenesis. Here, we provide evidence for an unexpected pro-microbial role of IRF3: the replication of the protozoan parasite, Toxoplasma gondii, was significantly impaired in IRF3-/- cells. In exploring whether the transcriptional activity of IRF3 was important for its pro-parasitic function, we found that ISGs induced by parasite-activated IRF3 were indeed essential, whereas type I interferons were not important. To delineate the signaling pathway that activates IRF3 in response to parasite infection, we used genetically modified human and mouse cells. The pro-parasitic signaling pathway, which we termed PISA (Parasite-IRF3 Signaling Activation), activated IRF3 without any involvement of the Toll-like receptor or RIG-I-like receptor pathways, thereby ruling out a role of parasite-derived RNA species in activating PISA. Instead, PISA needed the presence of cGAS, STING, TBK1 and IRF3, indicating the necessity of DNA-triggered signaling. To evaluate the physiological significance of our in vitro findings, IRF3-/- mice were challenged with parasite infection and their morbidity and mortality were measured. Unlike WT mice, the IRF3-/- mice did not support replication of the parasite and were resistant to pathogenesis caused by it. Our results revealed a new paradigm in which the antiviral host factor, IRF3, plays a cell-intrinsic pro-parasitic role.

The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity.

BACKGROUND: Interferon (IFN) inhibits viruses by inducing several hundred cellular genes, aptly named 'interferon (IFN)-stimulated genes' (ISGs). The only two RNA viruses of the Pneumovirus genus of the Paramyxoviridae family, namely Respiratory Syncytial Virus (RSV) and Pneumonia Virus of Mice (PVM), each encode two nonstructural (NS) proteins that share no sequence similarity but yet suppress IFN. Since suppression of IFN underlies the ability of these viruses to replicate in the host cells, the mechanism of such suppression has become an important area of research. This Short Report is an important extension of our previous efforts in defining this mechanism. RESULTS: We show that, like their PVM counterparts, the RSV NS proteins also target multiple members of the ISG family. While significantly extending the substrate repertoire of the RSV NS proteins, these results, unexpectedly, also reveal that the target preferences of the NS proteins of the two viruses are entirely different. This is surprising since the two Pneumoviruses are phylogenetically close with similar genome organization and gene function, and the NS proteins of both also serve as suppressors of host IFN response. CONCLUSION: The finding that the NS proteins of the two highly similar viruses suppress entirely different members of the ISG family raises intriguing questions of pneumoviral NS evolution and mechanism of action.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

Extraribosomal l13a is a specific innate immune factor for antiviral defense.

UNLABELLED: We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3'untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense. IMPORTANCE: The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3' untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity.

An extraribosomal function of ribosomal protein L13a in macrophages resolves inflammation.

Inflammation is an obligatory attempt of the immune system to protect the host from infections. However, unregulated synthesis of proinflammatory products can have detrimental effects. Although mechanisms that lead to inflammation are well appreciated, those that restrain it are not adequately understood. Creating macrophage-specific L13a-knockout mice, we report that depletion of ribosomal protein L13a abrogates the endogenous translation control of several chemokines in macrophages. Upon LPS-induced endotoxemia, these animals displayed symptoms of severe inflammation caused by widespread infiltration of macrophages in major organs causing tissue injury and reduced survival rates. Macrophages from these knockout animals show unregulated expression of several chemokines (e.g., CXCL13, CCL22, CCL8, and CCR3). These macrophages failed to show L13a-dependent RNA binding complex formation on target mRNAs. In addition, increased polyribosomal abundance of these mRNAs shows a defect in translation control in the macrophages. Thus, to our knowledge, our studies provide the first evidence of an essential extraribosomal function of ribosomal protein L13a in resolving physiological inflammation in a mammalian host.

Respiratory syncytial virus mechanisms to interfere with type 1 interferons.

Respiratory syncytial virus (RSV) is a member of the Paramyxoviridae family that consists of viruses with nonsegmented negative-strand RNA genome. Infection by these viruses triggers the innate antiviral response of the host, mainly type I interferon (IFN). Essentially all other viruses of this family produce IFN suppressor functions by co-transcriptional RNA editing. In contrast, RSV has evolved two unique nonstructural proteins, NS1 and NS2, to effectively serve this purpose. Together, NS1 and NS2 degrade or sequester multiple signaling proteins that affect both IFN induction and IFN effector functions. While the mechanism of action of NS1 and NS2 is a subject of active research, their effect on adaptive immunity is also being recognized. In this review, we discuss various aspects of NS1 and NS2 function with implications for vaccine design.