Benjamin R. tenOever

The interferon antiviral response: from viral invasion to evasion.

One of the initial responses of an organism to infection by pathogenic viruses is the synthesis of antiviral cytokines such as the type I interferons (interferon-α/β), interleukins, and other proinflammatory cytokines and chemokines. Interferons provide a first line of defence against virus infections by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response. Interferons stimulate cells in the local environment to activate a network of interferon-stimulated genes, which encode proteins that have antiviral, antiproliferative and immunomodulatory activities. The present review focuses on recent reports that describe the activation of multiple signalling pathways following virus infection, new candidate genes that are implicated in the establishment of the antiviral state, and the strategies used by viruses and their specific viral products to antagonize and evade the host antiviral response.

Recognition of the Measles Virus Nucleocapsid as a Mechanism of IRF-3 Activation

ABSTRACT The mechanisms of cellular recognition for virus infection remain poorly understood despite the wealth of information regarding the signaling events and transcriptional responses that ensue. Host cells respond to viral infection through the activation of multiple signaling cascades, including the activation of NF-κB, c-Jun/ATF-2 (AP-1), and the interferon regulatory factors (IRFs). Although viral products such as double-stranded RNA (dsRNA) and the processes of viral binding and fusion have been implicated in the activation of NF-κB and AP-1, the mechanism(s) of IRF-1, IRF-3, and IRF-7 activation has yet to be fully elucidated. Using recombinant measles virus (MeV) constructs, we now demonstrate that phosphorylation-dependent IRF-3 activation represents a novel cellular detection system that recognizes the MeV nucleocapsid structure. At low multiplicities of infection, IRF-3 activation is dependent on viral transcription, since UV cross-linking and a deficient MeV containing a truncated polymerase L gene failed to induce IRF-3 phosphorylation. Expression of the MeV nucleocapsid (N) protein, without the requirement for any additional viral proteins or the generation of dsRNA, was sufficient for IRF-3 activation. In addition, the nucleocapsid protein was found to associate with both IRF-3 and the IRF-3 virus-activated kinase, suggesting that it may aid in the colocalization of the kinase and the substrate. Altogether, this study suggests that IRF-3 recognizes nucleocapsid structures during the course of an MeV infection and triggers the induction of interferon production.

Activation of TBK1 and IKKε Kinases by Vesicular Stomatitis Virus Infection and the Role of Viral Ribonucleoprotein in the Development of Interferon Antiviral Immunity

ABSTRACT Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKε have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKε kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKε activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKε following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKε kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKε in epithelial cells. Induction of IKKε potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.

MicroRNA-mediated species-specific attenuation of influenza A virus.

Influenza A virus leads to yearly epidemics and sporadic pandemics. Present prophylactic strategies focus on egg-grown, live, attenuated influenza vaccines (LAIVs), in which attenuation is generated by conferring temperature sensitivity onto the virus. Here we describe an alternative approach to attenuating influenza A virus based on microRNA-mediated gene silencing. By incorporating nonavian microRNA response elements (MREs) into the open-reading frame of the viral nucleoprotein, we generate reassortant LAIVs for H1N1 and H5N1 that are attenuated in mice but not in eggs. MRE-based LAIVs show a greater than two-log reduction in mortality compared with control viruses lacking MREs and elicit a diverse antibody response. This approach might be combined with existing LAIVs to increase attenuation and improve vaccine safety.

RNA viruses and the host microRNA machinery

Gene silencing by small RNAs (sRNAs) occurs in all three domains of life. In recent years, our appreciation of the diverse functions of sRNAs has increased, and we have identified roles for these RNAs in cellular differentiation, fitness and pathogen defence. Interestingly, although plants, nematodes and arthropods use sRNAs to combat viral infections, chordates have replaced this defence strategy with one based exclusively on proteins. This limits chordate use of sRNAs to the silencing of genome-encoded transcripts and has resulted in viruses that do not perturb sRNA-related cellular processes. This evolutionary phenomenon provides an opportunity to exploit the pre-existing chordate sRNA pathways in order to generate a range of virus-based biological tools. Here, I discuss the relationship between sRNAs and RNA viruses, detail how microRNA expression can be harnessed to control RNA viruses and describe how RNA viruses can be designed to deliver sRNAs.

Noncanonical cytoplasmic processing of viral microRNAs

Cellular utilization of RNA interference (RNAi) as a mechanism to combat virus infection is thought to be restricted to plants and invertebrates. In vertebrates, antiviral defenses are largely dependent on interferons (IFNs), with the use of small RNAs restricted to microRNA (miRNA)-mediated targeting of host transcripts. Here we demonstrate that incorporation of a primary miRNA into a cytoplasmic virus results in the formation of a Dicer-dependent, DGCR8-independent, mature miRNA capable of conferring RNAi-like activity. Processing of the viral mirtron-like product (virtron) is indistinguishable from endogenous miRNA maturation and elicits post-transcriptional gene silencing, albeit at a reduced level. Furthermore, virtrons impose Dicer-dependent, microprocessor-independent, and IFN-independent interference on virus replication in a sequence-specific manner. Taken together, these results suggest the existence of a noncanonical, small-RNA-based activity capable of processing cytoplasmic hairpins and perhaps contributing to the cells antiviral arsenal.

Influenza A Virus Transmission Bottlenecks Are Defined by Infection Route and Recipient Host

Despite its global relevance, our understanding of how influenza A virus transmission impacts the overall population dynamics of this RNA virus remains incomplete. To define this dynamic, we inserted neutral barcodes into the influenza A virus genome to generate a population of viruses that can be individually tracked during transmission events. We find that physiological bottlenecks differ dramatically based on the infection route and level of adaptation required for efficient replication. Strong genetic pressures are responsible for bottlenecks during adaptation across different host species, whereas transmission between susceptible hosts results in bottlenecks that are not genetically driven and occur at the level of the recipient. Additionally, the infection route significantly influences the bottleneck stringency, with aerosol transmission imposing greater selection than direct contact. These transmission constraints have implications in understanding the global migration of virus populations and provide a clearer perspective on the emergence of pandemic strains.

Engineered RNA viral synthesis of microRNAs

MicroRNAs (miRNAs) are short noncoding RNAs that exert posttranscriptional gene silencing and regulate gene expression. In addition to the hundreds of conserved cellular miRNAs that have been identified, miRNAs of viral origin have been isolated and found to modulate both the viral life cycle and the cellular transcriptome. Thus far, detection of virus-derived miRNAs has been largely limited to DNA viruses, suggesting that RNA viruses may be unable to exploit this aspect of transcriptional regulation. Lack of RNA virus-produced miRNAs has been attributed to the replicative constraints that would incur following RNase III processing of a genomic hairpin. To ascertain whether the generation of viral miRNAs is limited to DNA viruses, we investigated whether influenza virus could be designed to deliver functional miRNAs without affecting replication. Here, we describe a modified influenza A virus that expresses cellular microRNA-124 (miR-124). Insertion of the miR-124 hairpin into an intron of the nuclear export protein transcript resulted in endogenous processing and functional miR-124. We demonstrate that a viral RNA genome incorporating a hairpin does not result in segment instability or miRNA-mediated genomic targeting, thereby permitting the virus to produce a miRNA without having a negative impact on viral replication. This work demonstrates that RNA viruses can produce functional miRNAs and suggests that this level of transcriptional regulation may extend beyond DNA viruses.

IκB kinase ε (IKKε) regulates the balance between type I and type II interferon responses

Virus infection induces the production of type I and type II interferons (IFN-I and IFN-II), cytokines that mediate the antiviral response. IFN-I (IFN-α and IFN-β) induces the assembly of IFN-stimulated gene factor 3 (ISGF3), a multimeric transcriptional activation complex composed of STAT1, STAT2, and IFN regulatory factor 9. IFN-II (IFN-γ) induces the homodimerization of STAT1 to form the gamma-activated factor (GAF) complex. ISGF3 and GAF bind specifically to unique regulatory DNA sequences located upstream of IFN-I– and IFN-II–inducible genes, respectively, and activate the expression of distinct sets of antiviral genes. The balance between type I and type II IFN pathways plays a critical role in orchestrating the innate and adaptive immune systems. Here, we show that the phosphorylation of STAT1 by IκB kinase epsilon (IKKε) inhibits STAT1 homodimerization, and thus assembly of GAF, but does not disrupt ISGF3 formation. Therefore, virus and/or IFN-I activation of IKKε suppresses GAF-dependent transcription and promotes ISGF3-dependent transcription. In the absence of IKKε, GAF-dependent transcription is enhanced at the expense of ISGF3-mediated transcription, rendering cells less resistant to infection. We conclude that IKKε plays a critical role in regulating the balance between the IFN-I and IFN-II signaling pathways.

Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals

While RNA interference (RNAi) functions as an antiviral response in plants, nematodes, and arthropods, a similar antiviral role in mammals has remained controversial. Three recent papers provide evidence that either favors or challenges this hypothesis. Here, we discuss these new findings in the context of previous research.