JVI Accepts, published online ahead of print on 28 May 2014 J. Virol. doi:10.1128/JVI.00707-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Defective viral genomes: critical danger signals of viral infections
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Carolina B. López1,*
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South University Avenue, Philadelphia, Pennsylvania 19104, USA.
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* Correspondence:
[email protected] Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380
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Viruses efficiently block the host antiviral response in order to replicate and spread before host
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intervention. The mechanism initiating antiviral immunity during stealth viral replication is
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unknown, but recent data demonstrate that defective viral genomes generated at peak virus
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replication are critical for this process in vivo. This article summarizes the supporting evidence
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and highlights gaps in our understanding of the mechanisms and impact of immunostimulatory
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defective viral genomes generated during natural infections.
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A BATTLE FOR VIRUS AND HOST COEXISTENCE
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To ensure survival, living organisms must recognize and counteract harmful invaders. In
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higher species, an army of proteins and cells has evolved to quickly and effectively eliminate
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viruses and other dangerous microbes. To prevent unnecessary damage to the host, this defense
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system is tightly controlled and only responds following recognition of pathogen associated
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molecular patterns (PAMPs). Viruses capable of escaping or counteracting the antiviral host
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response have an evolutionary advantage, as they can replicate to high titers and spread before
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being eradicated. Many examples of viruses that effectively counteract host defenses are found
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among seasonally circulating viruses. For example, influenza virus and respiratory syncytial
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virus encode proteins that antagonize the host response, allowing these viruses to replicate for
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1.5 - 3 days in the absence of host antiviral activity (7). This so called “incubation period” is
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characterized by rapid virus growth in the absence of symptoms, such as fever and malaise, and
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is followed by an effective antiviral response that controls the infection and clears the virus.
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Synthetic viral mimics and viruses unable to counteract the host immune response elicit
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rapid and strong antiviral immunity. These systems have allowed for the identification of both
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viral PAMPs and cellular proteins involved in the initiation of the antiviral response. However,
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while it is clear that additional factors are required to trigger a potent host response in the
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presence of viral antagonists, the specific signals that are utilized remain to be identified. In the
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following sections, I summarize evidence indicating that for many viruses bearing a negative
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sense RNA genome, those signals are provided by defective viral genomes (DVGs) that are
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generated as replication byproducts when the virus reaches high titers. The relatively late
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generation of DVGs in the virus infection cycle, coupled with their effective detection by host
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cellular proteins and the induction of a potent antiviral response, is a testament to the
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evolutionary compromise to sustain virus-host coexistence.
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ORIGIN AND ACTIVITY OF DVGs
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DVGs are truncated forms of the viral genome that are generated during virus replication
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at high titers. DVGs lack essential genes and cannot propagate in the absence of helper virus.
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Two major types of DVGs have been described for RNA viruses. Deletion DVGs are truncated
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versions of the original virus genome that normally share the 3’ and 5’ ends with the parental
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virus. They are generated when the viral polymerase falls off the original template and reattaches
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further downstream, resulting in a genomic deletion. Copy-back DVGs, and the related snap-
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back DVGs, consist of a segment of the viral genome flanked by reverse complementary
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versions of its 5’ end. Copy-back DVGs occur when the viral polymerase detaches from the
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template and reattaches to the newly synthesizing strand, copying back the 5’ end of the genome
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(reviewed in (2, 6)). In the absence of viral nucleoproteins, copy-back genomes can form a
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hairpin structure through interactions between their 5’ and 3’ ends. It remains to be demonstrated
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whether these structures are formed in the context of virus infection, as nascent RNA folds
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during synthesis and is quickly bound by virus nucleoproteins.
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DVGs of RNA viruses were first described for influenza virus in the late 1940s (8). Since
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then, they have been identified in a variety of viruses passaged at high multiplicity of infection in
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vitro (13). DVGs were initially characterized as the genomes of defective viral particles able to
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interfere with standard virus replication, hence earning the name of defective interfering (DI)
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particles (13). In the late 70s and early 80s a series of reports demonstrated that density-purified
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defective viral particles from vesicular stomatitis virus, Sindbis virus, Sendai virus (SeV), and
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other viruses strongly induced type I IFN expression during infection of cells in vitro (4, 5). The
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ability of DI particles to promote strong type I IFN induction was subsequently confirmed using
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recombinant viruses (15). The interfering activity and the type I IFN inducing potential led to the
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study of DI particles as vaccine adjuvants and as broad spectrum antivirals (reviewed in (2)).
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Although DI particles have not yet been tried as immunostimulatory molecules in the clinic,
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large amounts of DI particles were found in vaccine strains of poliovirus and measles virus (9,
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14).
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CELLULAR RESPONSE TO DVGs
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Using the mouse paramyxovirus pathogen SeV as a model, our group showed that in
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addition to promoting type I IFN expression, DI particles stimulate the rapid and strong
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expression of a large panel of pro-inflammatory cytokines, chemokines, antiviral genes, and cell
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surface molecules on mouse and human dendritic cells. DI particles rendered these cells fully
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capable of stimulating T cells, thereby promoting the transition from the innate to the adaptive
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immune response (10, 16, 18, 19). Stocks of SeV lacking DI particles failed to induce a potent
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response even at high multiplicity of infection, but potent activation of dendritic cells was
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restored following supplementation with purified DI particles. Remarkably, the stimulatory
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activity of DVGs in vitro was independent of protein secretion and was maintained in the
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absence of type I IFN signaling and in the presence of functional virus antagonists of the
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antiviral response (18, 19), supporting their potential ability to induce a host response during
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periods of stealth replication.
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RNA viruses are primarily recognized in infected cells by two intracellular helicases, the
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retinoic acid-inducible gene-1-like receptors (RLRs) retinoic acid-inducible gene-1 (RIG-I) and
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melanoma differentiation associated protein 5 (MDA5). The binding of viral PAMPs to the
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RNA-binding domain of RLRs triggers their phosphorylation, oligomerization, and signaling
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through the adaptor protein mitochondrial antiviral-signaling protein (MAVS) (reviewed in (3)).
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RLR signaling activates transcription factors that drive the expression of the major antiviral
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cytokines type I IFN, as well as of pro-inflammatory and antiviral proteins. A type I IFN
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signaling loop that amplifies the expression of many key molecules in these pathways, including
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RLRs themselves, maximizes the RLR response (3). Notably, many of the viral proteins that
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antagonize the host immune response directly target the RLR pathway. For example, the
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parainfluenza virus V protein directly blocks MDA5 signaling, while its C protein blocks type I
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IFN signaling and amplification of the RLR response; respiratory syncytial virus NS1 and NS2
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interfere with the expression and signaling of type I IFNs; and the influenza virus NS1 protein
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interferes with the RLR pathway at multiple levels (reviewed in (17)). Recombinant viruses
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lacking these antagonistic proteins induce a fast and potent immune response that results in rapid
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viral clearance (11), demonstrating that viral-encoded proteins inhibit early recognition of
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viruses by RLRs in vivo.
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Although the mechanism for the efficient and potent response to DVGs in the presence of
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virus antagonists has not been fully elucidated, data show that DVGs are recognized by RLRs in
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infected cells. In support, MAVS deficiency abrogates the generation of the antiviral response to
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DVGs (16). In addition, RIG-I binds DVGs preferentially over standard virus genomes in cells
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infected with SeV or influenza virus (1) and RIG-I overexpression enhances the response to SeV
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DVGs (19). MDA5 also participates in the response to DVGs and is essential for a fast response
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in vitro (18). Notably, the expression of MDA5 is amplified by DVGs independently of type I
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IFNs (20). Toll-like receptors, another major family of virus-sensing molecules, are dispensable
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for the recognition of DVGs in infected cells and it remains to be determined if they participate
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in DVG recognition during complex in vivo infections.
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IMMUNOSTIMULATORY MOLECULAR MOTIFS OF DVGs
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Studies using synthetic viral RNA analogs or purified viral RNA revealed that RIG-I
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binds to uncapped 5’di- or tri-phosphates (5’ppp) coupled to short dsRNA stretches or single
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stranded RNA, while MDA5 binds to long stretches of dsRNA and complex RNA secondary
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structures (reviewed in (3)). Although both deletion and copy-back DVGs preserve the RIG-I
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stimulatory uncapped 5’ppp motif, copy-back DVGs have significantly more potent
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immunostimulatory activity than other forms of the genome (15, 19). While the potent activity of
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DVGs may be partially explained by a faster accumulation in infected cells (16) due to their
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strong flanking antigenomic promoters, infection with large amounts of standard virus does not
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compensate for their poor immunostimulatory activity (16, 19), suggesting that the relative level
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of PAMPs does not fully account for the more potent recognition of DVGs. Interestingly,
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treatment of SeV DVGs with phosphatase to eliminate the 5’ppp motif, significantly diminishes
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but does not eliminate their immunostimulatory ability (10, 16), suggesting that other RNA
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motifs play a role in DVG recognition. Using in vitro transcribed RNA from a highly
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immunostimulatory SeV DVG, a critical role for secondary structures in the immunostimulatory
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activity of DVG RNA is beginning to emerge (10, 12).
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DVGs IN NATURAL INFECTIONS
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Although the accumulation of immunostimulatory DVGs in cells infected in vitro has
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been demonstrated for many viruses (4-6, 13), it remained unclear if DVGs have a fundamental
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biological role during natural infections. Early observations in infected cell lines suggested that
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the interplay between the interfering capacity of DI particles and the requirement for standard
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virus for DI expansion would lead to alternating cycles of standard virus genome and DI particle
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replication, thereby promoting virus persistence. However, evidence for a role for DI particles in
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promoting virus persistence in vivo is lacking. Historically, technical difficulties in
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differentiating standard and defective viral genomes, together with a wide spread belief that
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DVGs were an epiphenomenon of in vitro virus replication discouraged further investigation of
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DVGs in natural infections. However, as DVGs that have a strong immunostimulatory activtiy
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have been reported in sera from patients infected with various viruses including hepatitis B virus,
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dengue, and influenza virus (reviewed in (2)), we set out to investigate potential roles for DVGs
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in the virus-host interaction during natural infections. Using SeV and mouse-adapted influenza
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virus, we demonstrated that DVGs are first detected in the lung at the time of peak virus
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replication (16), coincident with the first signs of expression of type I IFNs and other host
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antiviral effector molecules. Importantly, DVGs were detected in mice lacking the type I IFN
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receptor, indicating that their generation is not driven by type I IFNs. Strikingly, lung cells
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containing high levels of standard virus genomes, but not DVGs, failed to express type I IFNs,
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while those containing both standard virus genomes and DVGs expressed type I IFN (16).
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Altogether, these data are the first demonstration of a pivotal role for DVGs in determining the
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onset of the antiviral immune response in natural infections.
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CONTRIBUTIONS TO THE FIELD AND FUTURE DIRECTIONS
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Technological advances and a new understanding of the molecular mechanisms for virus
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recognition in infected cells have revived the interest in studying the biology and
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immunostimulatory function of DVGs. Our recent demonstration that DVG accumulation during
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infections in vivo stimulates a potent antiviral response (16) proves that these ubiquitous
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byproducts of virus replication play a pivotal role in natural virus-host interactions.
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A number of knowledge gaps in our understanding of immunostimulatory DVGs still
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exist. It is unclear whether DVG-mediated interference with standard virus replication is required
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for potent induction of antiviral activity or if interfering and immunostimulatory DVGs can be
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distinct. The molecular mechanisms driving the generation of DVGs are yet unknown and it
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remains to be determined if they arise as a consequence of the error prone viral RNA polymerase
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that allows for the generation of quasispecies essential to maintain virus fitness, or if distinct
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mechanisms drive DVG generation. The cellular pathways and secondary RNA motifs involved
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in the efficient recognition and potent response to DVGs in the presence of functional virus
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antagonists remain poorly understood, and is likely that novel circuits that modulate the function
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of RLRs are involved. Importantly, the role of DVGs in determining virus pathogenesis remains
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to be studied. Vigorous research on the mechanisms and impact of immunostimulatory DVGs in
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various viral infections will fill these gaps of knowledge and open novel opportunities for
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harnessing the DVG-host interactions for therapeutic intervention.
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ACKNOWLEDGEMENTS
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I thank all past and present members of my laboratory for their hard work and valuable
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contributions to the study of defective viral genomes. I thank Debora Argento for help with the
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figure and Leslie King and Susan Weiss for critically reading the manuscript. I apologize to all
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colleagues whose work could not be referenced because of length restrictions.
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Work in our laboratory is currently supported by grants from the NIH AI083284 and The
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University of Pennsylvania Research Foundation.
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Figure Legend
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effective antiviral responses to infections with paramyxoviruses: (A) According to current
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paradigms, viral recognition is maximized by type I IFN signaling that promotes the
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expression of essential viral sensors and signaling molecules. This paradigm assumes a low
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level of expression of type I IFN. In the depicted example of SeV infection, this scenario is
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possible if the viral encoded V and C antagonistic proteins are only partially active, for
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example during a zoonotic infection. However, this scenario cannot explain the induction of
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the antiviral response in the natural host of this virus, which is effectively blocked by the
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viral antagonists. (B) Our model proposes that defective viral genomes that arise at high
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levels of virus replication provide potent danger signals that stimulate the host antiviral
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response overcoming viral antagonism and independent of type I IFN feedback.
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Figure 1. Current and proposed models of the events required for the development of