Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells.

IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.

Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

Modular cell-based platform for high throughput identification of compounds that inhibit a viral interferon antagonist of choice.

Viral interferon (IFN) antagonists are a diverse class of viral proteins that counteract the host IFN response, which is important for controlling viral infections. Viral IFN antagonists are often multifunctional proteins that perform vital roles in virus replication beyond IFN antagonism. The critical importance of viral IFN antagonists is highlighted by the fact that almost all viruses encode one of these proteins. Inhibition of viral IFN antagonists has the potential to exert pleiotropic antiviral effects and thus this important protein class represents a diverse plethora of novel therapeutic targets. To exploit this, we have successfully developed and executed a novel modular cell-based platform that facilitates the safe and rapid screening for inhibitors of a viral IFN antagonist of choice. The platform is based on two reporter cell-lines that provide a simple method to detect activation of IFN induction or signaling via an eGFP gene placed under the control of the IFNß or an ISRE-containing promoter, respectively. Expression of a target IFN antagonist in the appropriate reporter cell-line will block the IFN response and hence eGFP expression. We hypothesized that addition of a compound that inhibits IFN antagonist function will release the block imposed on the IFN response and hence restore eGFP expression, providing a measurable parameter for high throughput screening (HTS). We demonstrate assay proof-of-concept by (i) exploiting hepatitis C virus (HCV) protease inhibitors to inhibit NS3-4A's capacity to block IFN induction and (ii) successfully executing two HTS targeting viral IFN antagonists that block IFN signaling; NS2 and IE1 from human respiratory syncytial virus (RSV) and cytomegalovirus (CMV) respectively, two clinically important viruses for which vaccine development has thus far been unsuccessful and new antivirals are required. Both screens performed robustly and Z' Factor scores of >0.6 were achieved. We identified (i) four hit compounds that specifically inhibit RSV NS2's ability to block IFN signaling by mediating STAT2 degradation and exhibit modest antiviral activity and (ii) two hit compounds that interfere with IE1 transcription and significantly impair CMV replication. Overall, we demonstrate assay proof-of-concept as we target viral IFN antagonists from unrelated viruses and demonstrate its suitability for HTS.

Targeting Pattern Recognition Receptors (PRR) for Vaccine Adjuvantation: From Synthetic PRR Agonists to the Potential of Defective Interfering Particles of Viruses.

Modern vaccinology has increasingly focused on non-living vaccines, which are more stable than live-attenuated vaccines but often show limited immunogenicity. Immunostimulatory substances, known as adjuvants, are traditionally used to increase the magnitude of protective adaptive immunity in response to a pathogen-associated antigen. Recently developed adjuvants often include substances that stimulate pattern recognition receptors (PRRs), essential components of innate immunity required for the activation of antigen-presenting cells (APCs), which serve as a bridge between innate and adaptive immunity. Nearly all PRRs are potential targets for adjuvants. Given the recent success of toll-like receptor (TLR) agonists in vaccine development, molecules with similar, but additional, immunostimulatory activity, such as defective interfering particles (DIPs) of viruses, represent attractive candidates for vaccine adjuvants. This review outlines some of the recent advances in vaccine development related to the use of TLR agonists, summarizes the current knowledge regarding DIP immunogenicity, and discusses the potential applications of DIPs in vaccine adjuvantation.

Identification of Novel Inhibitors of the Type I Interferon Induction Pathway Using Cell-Based High-Throughput Screening.

Production of type I interferon (IFN) is an essential component of the innate immune response against invading pathogens. However, its production must be tightly regulated to avoid harmful effects. Compounds that modulate the IFN response are potentially valuable for a variety of applications due to IFN's beneficial and detrimental roles. We developed and executed a cell-based high-throughput screen (HTS) targeting components that participate in and/or regulate the IRF3 and nuclear factor (NF)-κB branches of the IFN induction pathway. The assay detects activation of the IFN induction pathway via an enhanced green fluorescent protein (eGFP) reporter gene under the control of the IFNß promoter and was optimized, miniaturized, and demonstrated suitable for HTS as robust Z' factor scores of >0.6 were consistently achieved. A diversity screening set of 15,667 small molecules was assayed and two novel hit compounds validated that specifically inhibit the IFN induction pathway. We demonstrate that one of these compounds acts at or upstream of IRF3 phosphorylation. A second cell-based assay to detect activation of the IFN signaling (Jak-Stat) pathway via an eGFP reporter gene under the control of an IFN-stimulated response element (ISRE) containing MxA promoter also performed well (robust Z' factor >0.7) and may therefore be similarly used to identify small molecules that modulate the IFN signaling pathway.