Defective Interfering Particles of Negative-Strand RNA Viruses

Abstract

Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While DIPs have long been recognized, their exact contribution to the outcome of acute or persistent viral infections has remained elusive. Recent studies have identified defective viral genomes (DVGs) in human infections, including respiratory syncytial virus and influenza, and growing evidence indicates that DVGs influence disease severity and may contribute to viral persistence. Further, several studies have advanced our understanding of key viral and host factors that regulate DIP formation and activity. Here we review these discoveries and highlight key questions moving forward.

An Interfering Activity in Viral Cultures

In the mid-20th century, von Magnus discovered an interference phenomenon while culturing influenza virus [1]. Specifically, following high multiplicity infections, large quantities of ‘incomplete’ influenza virus particles were produced that interfered with the production of standard infectious virus particles (see Glossary) [1]. In the ensuing years, interfering particles were found to be produced by several RNA viruses including Rift Valley fever virus [2], vesicular stomatitis virus (VSV) [3], lymphocytic choriomeningitis virus (LCMV) [4], and Sendai virus (SeV) [5]. Importantly, it was shown that these interfering particles mediate homologous interference, but not heterologous interference [6]. For example, interfering particles from VSV did not interfere with the heterologous encephalomyocarditis virus, demonstrating that this interference phenomenon was not simply caused by a soluble antiviral molecule, such as interferon [7]. Recognizing this growing body of literature, Alice Huang and David Baltimore proposed that these particles may be a critical determinant of infection outcome for many viruses and coined the term defective interfering particles (DIPs) in a 1970 review [6]. In the next decade and a half, a flurry of activity significantly advanced this field of research. Seminal findings included the demonstration that DIPs correlated with the establishment of persistent in vitro infections, that DIPs could be isolated from experimentally infected animals, and that purified DIPs administered separately from infectious virus could protect animals from infectious virus-induced disease or death [8]. Despite these advances, evidence supporting a role for DIPs during the course of naturally acquired infections remained elusive. Investigations were hindered by the inability to prevent DIP-forming viruses from making DIPs during the course of in vivo infections and the lack of next-generation sequencing and reverse genetics technologies. Only more recently, armed with these tools, has the field begun to provide strong evidence to suggest that DIPs can influence the outcome of natural infections. Further, we now have a deeper understanding of the mechanisms by which DIPs interfere with standard infectious virus propagation and virus–host interactions that regulate DIP production. While DIPs have been described for a wide range of viruses, including DNA viruses, retroviruses, and positive-strand RNA viruses, here we focus on recent discoveries concerning DIPs produced by enveloped, negative-strand RNA viruses. Recent reviews on DIPs by other groups have covered topics not addressed here and readers may find those articles a useful complement [9–12].

Properties of DIPs

DIPs are virus particles released from cells that are biochemically and morphologically similar to standard virus particles but, in most instances, have been shown to harbor deletions in their genomes [13,14]. DIPs containing such defective viral genomes (DVGs) can often be readily separated by centrifugation, as with VSV DIPs, which are physically shorter than the distinct, bullet-shaped standard virus particles [15]. However, DIPs from viruses not known to contain large deletions in the genomes of their DIPs, like arenaviruses, cannot be separated from standard virus particles by gradient centrifugation [16]. Since DIPs contain DVGs, they are incapable of expressing the full complement of viral proteins that standard virus particles with full-length genomes express [14]. However, the ability to be replicated is critical for a DVG’s capacity to interfere with the production of standard virus [17]. While there is significant heterogeneity in the size, type, and location of deletions in DVGs, they must retain the replication initiation and termination elements that allow them to be replicated by the viral polymerase [17].

DIPs that package DVGs interfere with the production of standard virus through a highly specific, genome-mediated mechanism. This homologous interference is exquisitely specific: even different serotypes of the same virus species only exhibit partial homologous interference [7]. Various studies have demonstrated that DIPs do not interfere with standard virus by simply binding all the viral receptors on the surface of cells. Rather, DIPs interfere within the cell, as DIP-mediated interference can still occur if DIPs are added to a culture after standard virus [7,18]. RNA virus genome replication is fundamentally an error-prone process and the several classes of DVGs that exist are thought to arise as aberrant replication products produced when the viral polymerase, without release of the nascent strand, jumps to a distal location on either the template strand or the nascent strand itself, yielding an incomplete genome (reviewed in [17]). Viruses possess an intrinsic ability to generate DIPs, as even stocks of clonally pure standard virus, such as plaque-purified virus, will generate DIPs in the normal course of propagation [19]. A DVG, once formed, can be replicated by the viral polymerase and packaged into virus particles, forming DIPs, which can then enter new cells. If DIPs and standard virus particles infect the same cell, the standard virus will act as a ‘helper’ virus and DIP production will predominate (Figure 1). This replicative advantage of DIPs was originally thought to be mediated by the shorter length of DVGs [6,17], but increased promoter strength [20–23] and packaging efficiency [24,25] in certain DVGs are more likely mechanisms. DIPs, by definition, are unable to self-replicate, meaning that if a cell is only infected by DIPs, no virus particles will be produced [6].

Figure 1. A Model for the Generation of Defective Interfering Particles and the Outcomes of Infection of a Cell with Standard Virus Particles, Defective Interfering Particles, or Both.

A host cell infected with a standard virus particle will initially produce primarily standard infectious particles. Later, following the de novo generation of defective viral genomes, defective interfering particles will be produced. As defective genomes have a replicative advantage over standard genomes, they will begin to outnumber standard virus genomes. This will result in the cell producing primarily defective interfering particles. Once a defective interfering particle is released, it can infect other cells. A cell that is infected only by a defective interfering particle will presumably not produce defective interfering or standard virus particles. However, if an uninfected cell is infected with a standard infectious particle and a defective interfering particle, the defective interfering particle will block the production of new infectious particles. However, additional defective interfering particles may be produced in this setting.

The molecular basis by which some DIPs interfere with standard infectious virus propagation is not known. Notably, a substantial body of work has accumulated on arenavirus DIPs. For example, LCMV, which exhibits the majority of properties that define DIPs, does not produce classic DI genomes containing large internal deletions within open reading frames. Sanger sequencing efforts in the 1990s revealed that small deletions (2–40 nucleotides) in the 3′ and 5′ untranslated regions of the LCMV genome accumulate during acute and persistent infection (presumably at the same time DIPs accumulate), both in vitro and in vivo [26,27]. However, it is currently unknown whether these candidate DVGs can interfere with standard virus replication. Thus, there is an opportunity to apply next-generation sequencing and reverse genetics to map candidate DVGs and test whether they are truly the cause of interference by LCMV DIPs or whether a genome-independent mechanism may be at work [28].

Technological Advances Enabling Progress in DIP Research

A notable challenge in the study of DIPs has been the limited sensitivity of assays to either measure them directly or alternatively to measure their interfering activity. The interference caused by DIPs in a virus culture can be observed with some viruses when titering by plaque assay, particularly for viruses that produce high levels of DIPs, like LCMV (Figure 2) [28]. In wells inoculated with a high concentration of virus, little or no cytopathic effect (CPE; i.e., plaque formation) is observed. Only after the sample is diluted sufficiently (presumably so that infectious virus particles can enter a cell without an accompanying DIP) can CPE and countable plaques be discerned. While qualitatively useful, more precise assays are required to accurately quantitate DIPs. Historically, DIPs were quantified indirectly by combining the sample of interest containing DIPs and a constant amount of standard virus and inoculating this mixture onto cells followed by measurement of a reduction in standard virus yield (i.e., release of standard virus into supernatant) [3] or plaque formation [16]. These assays generally lacked great sensitivity, which limited the study of DIPs, particularly in vivo, where titers were often low [14,29,30]. However, for high DIP producing viruses, they still represent a valuable tool.

Figure 2. DIP-Mediated Interference with Infectious Virus Propagation Can Be Visualized by Plaque Assay.

Pictured is a representative plaque assay performed on a laboratory stock of LCMV strain Armstrong 53b. The highest concentration of infectious virus was added to the far left well and the wells to the right received successive serial 10-fold dilutions of the initial inoculum. Each well was stained with crystal violet and the image converted to gray scale (i.e., black staining indicates intact areas of the Vero E6 monolayer, while white areas/foci represent LCMV plaques). Starting from the left, note that despite receiving the most infectious virus, the first three dilutions show no or few plaques. This is presumably because all of the available cells were infected with DIPs that blocked propagation of any standard infectious particles that also infected those cells. Only by the fourth and fifth serial dilutions are individual plaques visible, presumably because DIPs were sufficiently diluted to allow standard infectious particles to productively infect cells in the absence of DIPs and thus create plaques. Abbreviations: DIPs, defective interfering particles; LCMV, lymphocytic choriomeningitis virus.

Several major technological advances since the late 1980s have enabled investigators to break new ground in the characterization of DIPs. The advent of PCR occurred in the mid-1980s [31], but a PCR assay to selectively amplify certain DVGs was not described until 1992 [32]. Such DVG-specific PCR assays have been subsequently employed to directly detect DVGs in human infections of measles virus (MeV) [33] and respiratory syncytial virus (RSV) [34]. DVG-specific PCR assays, however, require at least some sequence information about the DVGs themselves and often cannot detect the full range of DVGs that are present in a sample [35]. Modern next-generation sequencing technologies are changing this landscape and recently have been used to characterize DVGs found in several paramyxoviruses [35–40], influenza viruses [38,41–43], VSV [44], and flock house virus [45,46]. Next-generation sequencing has also permitted characterization of DVGs from clinical isolates of influenza patients [41,43,47], ultimately leading to some of the most compelling evidence that DVGs may influence the outcome of natural infections [41]. While these modern approaches for measuring DVGs are highly sensitive, they do not directly measure the interfering capacity of these genomes. It remains important for the field to demonstrate that presumptive DVGs uncovered by PCR and/or sequencing are (i) defective for permitting the full viral life cycle to be completed and (ii) able to interfere with standard virus propagation, which could be measured by classic assays such as the yield reduction assay described earlier. This validation of candidate DVGs is critical in cases where accumulation of these DVGs is being correlated with a particular outcome of infection.

In addition to sensitive detection of DVGs at the cell population level, technological advances have aided the analysis of DVGs at the single-cell level. High multiplicity infections can result in cell protection, less efficient standard virus production, and enhanced DIP production [48]. The theory behind this holds that during high multiplicity infections, a large proportion of cells will be infected with both infectious viruses and DIPs. As DIPs can inhibit standard virus production with single-hit kinetics, a single DIP infecting a cell may be enough to completely inhibit standard virus production in that cell and protect it from death [49–51]. Conversely, in low multiplicity infections, cells may be infected with DIPs only, standard virus only, or both. While this model has existed since Huang and Baltimore first proposed it [6], only recently has substantial evidence supporting the heterogeneous nature of infection in a population of cells begun to accumulate. Fluorescent reporter viruses and RNA fluorescence in situ hybridization coupled with fluorescence microscopy or flow cytometry have been used to monitor the spread of DIPs and standard virus during infection at the single cell level [52–54]. These technologies have provided mechanistic insight into how DIP-infected cells, despite the ability of some DVGs to potently activate the innate antiviral response, are able to selectively elicit a prosurvival pathway and escape death [54]. Furthermore, these technologies have shown that SeV DVGs fail to interact with host trafficking proteins and consequently, little or no DIPs are produced from cells with high levels of DVGs [53]. Collectively, this recent work has allowed a deeper mechanistic understanding of the dynamic interplay between DIPs and standard virus.

A significant barrier to determining the role of DIPs during viral infections has been the difficulty in separating the function of standard virus and DIPs. Viruses have a natural propensity to produce DIPs, meaning that even when starting with clonally pure standard infectious virus, DIPs will eventually be made. Several approaches have been used to circumvent this problem to the extent possible. Functionally pure DIPs can be obtained by irradiation with UV light for viruses with DIPs that are less sensitive to UV light relative to standard virus, like arenaviruses and influenza A virus (IAV) [16,55]. Differential centrifugation can be used for viruses like VSV in which the DIPs were different enough in size compared with standard virus [15]. DIP-enriched virus preparations can also be derived from persistently infected cells or from the acute phase of infection in cells that were inoculated at a high multiplicity of infection [15]. The ability to add DIPs of a certain genome type to cultures or to manipulate elements of the viral genome in order to selectively control DIP production was a feat that required reverse genetics systems. As an example, VSV DVGs were the first to be successfully generated from cloned cDNA and packaged into ‘infectious’ DIPs [56,57]. The development of reverse genetics systems for other negative-strand viruses [58] provided tools to help determine elements of viruses that contribute to the production of DIPs as will be discussed in the following sections.

Regulation of DIP Production by Viral and Host Factors

While DIPs accumulate during a wide range of viral infections, both viral and host factors can regulate their formation (see Table 1, Key Table, for a summary). Accumulation of DIPs is presumably a combined result of the de novo generation of new DVGs from the wild-type parental genome followed by the subsequent replication and incorporation of these DVGs into new virus particles. Replication of RNA virus genomes is inherently an error-prone process. Accordingly, de novo generation of DVGs is often described as a result of this error-prone process. However, recent evidence has demonstrated that viruses can reproducibly produce DVGs with defined sequences, suggesting that DVG generation can be directed and is not exclusively a product of random replication errors [59]. Identification of specific sequences in the genome of RSV that regulate the formation of copy-back DVGs has enabled the engineering of viruses that do not produce specific DVGs [59].

Table 1.

Viral and Host Factors That Regulate the Formation of DIPs

Virus	Genomic or protein level	Host or viral factor	De novo generation, replication, or packaging	Proposed mechanism ofaction	Refs
Lymphocytic choriomeningitis virus	Protein	Viral: Z matrix protein	Unknown	The PPXY late domain in the viral Z protein promotes DIP production	[28]
Lymphocytic choriomeningitis virus	Not applicable (n/a)	Host: ESCRT, host kinases, Nedd4 family E3 ubiquitin ligases	Unknown	Thecellular ESCRT pathway, host kinase-driven phosphorylation of the viral matrix protein, and certain Nedd4 family E3 ubiquitin ligases promote DIP production	[28,78,79]
Respiratory syncytial virus	Genomic	Viral	De novo	Nucleotide composition at specific sites in the genomic RNA increases the rate at which the viral polymerase generates copy-back DVGs	[59]
Influenza A virus	Unkown	Viral	Unknown	A mutation in the PA subunit in the polymerase results in a significant increase in DIPs. Function could be at the genomic level as the mutation in PA results in increased levels of DVGs with mutations in genome segment containing PA. The mutation could also affect the ability of PA to stay bound to the RNA template which would increase the chances of aberrant RNA replication	[74]
Influenza A virus	Protein	Viral	De novo	Mutations in NS2 increase generation of DIPs specifically containing deletions in PA gene	[69]
Vesicular stomatitis virus	n/a	Host	De novo	Human chromosome 16 in human–mouse somatic cell hybrids suppressed de novo generation of DVGs	[75]
Rabies virus	Genomic	Virus	Replication	Promoter strength of an engineered copy-back DVG permitted strongly biased amplification of the DVG over full-length genomes	[65]
Influenza A virus	Genomic	Viral	n/a packaging	IAV DVGs are packaged into virus particles more efficiently than their cognate full-length genomes	[24,66,67]
Influenza A virus	Genomic	Viral	Replication	A specific type of IAV DVGs has a mutation in its replication promoter that substantially increases the rate of replication relative to wild-type genomes	[61]
Vesicular stomatitis virus, Sendai virus	Genomic	Viral	Replication	Complementary ends and a lack of a transcriptional promoter allow 5′ copy-back and snap-back DVGs to replicate at a higher rate than full-length genomes	[61–64]
Influenza A virus	Likely Protein	Viral	Unknown	A mutation (D529N) in the PA subunit of the polymerase inhibited the production of DVGs	[41]
Influenza A virus	Likely Protein	Viral	Unknown	Mutations in M1 and M2 increase the production of DVGs	[41,73]
Sendai virus, measles virus	Likely protein	Viral	Unknown	The C protein suppresses DVG production	[36,70,71]
Sendai virus	Protein	Viral	De novo	A point mutation in the N protein increases production of DVGs, possibly by the lower density of viral nucleocapsids	[68]
Influenza A virus	Protein	Viral	Unknown	The NS gene segment from a highly pathogenic H5N1 IAV induces robust production of DIPs compared with the NS gene segment from H1N1	[70]

A DVG, once generated, typically will replicate at a much higher rate than full-length genomes. Huang and Baltimore originally proposed that this replicative advantage was a product of the shorter length of DVGs [6]; however, evidence has accumulated showing that factors other than the length of the genome may also contribute to this increase in the replicative capacity of DVGs. For example, a full-length, but defective interfering genome found in IAV can replicate more efficiently due to its increased promoter strength [60]. In addition, evidence has demonstrated that copy-back DVGs possess a significant replicative advantage by way of their terminal complementarity and, for 5′ copy-back DVGs, the lack of a transcriptional promoter [61–65]. Beyond de novo generation and replication, other factors may also influence the accumulation of DIPs. For example, IAV DVGs can be packaged more efficiently than their cognate full-length genomes [24,66,67].

Several viral proteins have been shown to regulate the de novo generation of DVGs. For example, specific mutations in the IAV NS2 protein and the SeV N protein increase DVG production [68,69] while the C protein of both SeV and MeV suppress DVG synthesis [36,70,71]. In addition, the IAV NS protein from the highly pathogenic H5N1 virus strongly drives the production of DIPs while NS from H1N1 does not [72]. Further, the IAV proteins M1 and M2 and the PA subunit of the polymerase also appear to regulate DIP production [41,73,74]. The production of DIPs is not only regulated by viral factors, but also by the cellular host. It has been shown that certain cell types produce little or no DIPs [14]. Interestingly, the de novo generation of DVGs is restricted in certain cell types [75–77]. However, the underlying mechanism(s) for these restrictions are largely unknown.

Our group recently identified both viral and host factors that regulate the formation of LCMV DIPs. Specifically, the PPXY late domain encoded by the LCMV matrix protein and the cellular ESCRT (endosomal sorting complexes required for transport) pathway, which is recruited by this late domain, are required for DIP production, but not standard infectious virus particles [28]. Further, certain host Nedd4 family E3 ubiquitin ligases, which bind to PPXY late domains, also specifically promote DIP production [78]. Last, phosphorylation of the LCMV matrix protein at multiple sites, including the PPXY late domain, appears to drive DIP formation, suggesting that arenaviruses can dynamically adjust DIP production in response to external environmental factors [28,79]. Collectively, these findings suggest that divergent pathways and regulatory mechanisms exist for the formation of standard particles versus DIPs in the setting of arenavirus infection.

DIPs in Natural Infections

Determining the role of DIPs during natural infections has been a major challenge and evidence that they can dictate infection outcome has only slowly emerged. The interference mediated by DIPs was first associated with ‘incomplete’ virus in the 1950s using experimental infections with Rift Valley fever virus and IAV in mice [2,80]. However, it took nearly two decades before the presence of DIPs from VSV [81], rabies virus [81], and LCMV [82] was confirmed in experimental infections in mice. In addition to their production in vivo, exogenous DIPs have been shown to reduce the pathology or delay or prevent death associated with homologous virus challenge in animals [8]. Arguably, some of the earliest studies of DIPs in natural infections were with LCMV, which is carried in nature by the common house mouse [82,83]. In addition, during a lethal outbreak of IAV H5N2 in chickens in the 1980s, it was found that the virulent strain failed to produce detectable DVGs while an avirulent strain did, suggesting that DIPs were a virulence factor [84,85]. DVGs have also been found in human infections, notably in brain tissue of humans with subacute sclerosing panencephalitis (SSPE) associated with MeV [86] and more recently in patients with IAV [41,43,47] and RSV [34] infections.

Evidence has also emerged that DIPs are not only present during natural infections, but can also help determine the course of disease. A clinical isolate of IAV H1N1 from a fatal case was identified that produced few DVGs while an isolate from a mild case of disease produced high levels of DVGs [41]. The specific genetic differences between the isolates that controlled DVG levels were identified, making it possible to recover recombinant viruses with genetic control over DVG production [41]. The high levels of DVGs produced by the ‘mild’ virus elicited an innate immune response in mice that helped control viremia and prevented neutrophil invasion and excessive inflammation that are associated with more severe disease [41]. In addition, in other IAV clinical isolates, increased levels of DVGs were associated with mild disease whereas clinical isolates from fatal cases had low levels of DVGs [41]. Similarly, detection of DVGs in the respiratory tract of children infected with RSV correlated with the expression of antiviral genes [34]. This work builds on in vitro studies showing that VSV DIPs activate the interferon response [87] and more recent studies demonstrating the critical role DVGs have in stimulating the innate immune response during several acute viral infections (for more a more detailed review of the immune response to DVGs, see [88]). DVGs are recognized by pattern recognition receptors, including retinoic acidinducible gene 1 (RIG-I), resulting in the production of interferon and other proinflammatory cytokines as well as the maturation of dendritic cells [9,89]. Specifically, copy-back or snapback DVGs for the nonsegmented, negative-strand RNA viruses or short deletion mutants in IAV have been implicated in activating antiviral innate immunity [14,38]. Indeed, the DVGs of these viruses, rather than standard virus genomes, are the primary activators of the interferon response [88]. The innate immune response activated by DVGs, at least in some cases, confers protection to the cells containing them by specifically activating a prosurvival pathway [54]. While this cellsparing activity initiated by the DVGs was first shown in 1977 for VSV [87], it is only recently that the signaling pathway through which interferon activation by DVGs results in cell sparing has been determined [54]. Collectively, DIPs appear to confer protection to their host by inhibiting standard virus production and/or activating the innate immune response resulting in a broad range of downstream consequences, but additional mechanisms may be at work.

DIPs in Persistent Infections

Viruses that establish persistent infections in their hosts must balance their own replication with the potential damage it may cause. Some non-retroviral RNA viruses are capable of establishing and maintaining persistent infections even though they cannot integrate their genomes into host chromosomes, like retroviruses, or maintain their genomes as episomes, like DNA viruses [90–92]. Huang and Baltimore originally proposed that DIPs may be a critical factor during persistent viral infections [6]. DIPs have been studied in the context of persistent infections in vitro with several viruses and are characterized by cyclical infections in which the levels of DIPs and standard virus rise and fall in alternating patterns [14,48]. There is significantly less known about DIPs and persistent infections in vivo. LCMV infection in mice is a particularly relevant model as LCMV is normally maintained in nature through persistent, asymptomatic infections in Mus musculus [93]. DIPs have been found in persistent LCMV infections in mice and are suspected to function during these infections [82], though numerous other factors are likely involved [94]. It has been particularly difficult to determine how DIPs dictate the course of persistent infections given the intrinsic ability of standard virus to generate DIPs, resulting in a mixture of DIPs and standard viruses during infection. In the case of LCMV, we have now engineered viruses that no longer produce DIPs (but still make normal levels of standard virus particles) [28,78], which opens the door to exploring their impact on the establishment and maintenance of an asymptomatic, persistent infection.

There has also been substantial interest in whether DIPs contribute to viral persistence in human infections including in Ebola virus and MeV infections, which are typically acute in humans, but are increasingly suspected to cause persistent infections. DIPs have been found in the persistent manifestation of MeV infection in the brain, SSPE, and it has been suggested that DIPs may play a role in this disease by helping to establish or maintain persist infection which can lead to SSPE [33,92]. For Ebola virus, during the unprecedented outbreak in 2014, some survivors harbored active infections long after resolution of acute disease [95,96], which may facilitate sexual transmission [97], or development of neurological disease [98]. It has been suggested that DIPs may have a role in this persistent state [99], as Ebola virus DIPs have been detected during in vitro infections [100], but to date it is unclear whether these patients harbor significant levels of DIPs [101].

Concluding Remarks

The collective understanding of this interfering phenomenon from ‘incomplete’ virus has come a long way in the past 70 years (see Figure 3 for a timeline of notable discoveries) but several key questions remain to be answered (see Outstanding Questions). For example, it remains to be determined whether DIPs function in a broader range of natural infections and the various mechanisms underlying their function. At present, little is known about viral and host factors that regulate the production of DIPs, but progress can certainly be made in this area. An improved understanding of these factors could provide strategies for transforming virulent viruses into live-attenuated vaccines by enhancing their capacity to produce DIPs. The mechanisms underlying persistent infections could also potentially be advanced if the tools that have been used to study DIPs in acute viral infections were applied. Finally, many important viral pathogens are zoonotic and are carried by their hosts persistently (e.g., orthohantaviruses and mammarenaviruses). Understanding the role that DIPs may have in the maintenance of these viruses in nature will be critical for understanding virus–host ecology and transmission dynamics.

Figure 3. Timeline of Notable Defective Interfering Particle Discoveries. See also [1,6–8,13,28,32,34,38,41,43,54,56,58,59,78,79,82,86].

Abbreviations: DIPs, defective interfering particles; DVGs, defective viral genomes; FISH, fluorescence in situ hybridization; IAV, influenza A virus; LCMV, lymphocytic choriomeningitis virus; MeV, measles virus; RSV, respiratory syncytial virus; SSPE, subacute sclerosing panencephalitis; VSV, vesicular stomatitis virus.

Outstanding Questions.

Are DIPs a general virulence factor for many viruses in the setting of human infection? If so, could detection and/or quantitation of DIPs inform treatment decisions?

What role do DIPs play in the setting of persistent viral infection of sylvatic reservoir species (i.e., mammarenavirus or orthohantavirus infection of rodents)? Are they critical for protection of host fitness and thus viral maintenance in nature? What is their role during persistent infection in humans (i.e., Ebola or Lassa viruses)?

What is the molecular basis of interference employed by DIPs produced by mammarenaviruses and orthohantaviruses? Is it DVG independent? If not, what classes of DVGs are produced and by what molecular mechanism?

How important are DIPs for the development and durability of protective immunity, particularly antiviral memory B and T cells?

What are the different viral and host factors that regulate DIP production for different viruses? How do these factors mechanistically drive or repress DIP production?

Can mechanistic knowledge of the virus and host factors involved in DIP production be harnessed to provide better treatment for acutely ill individuals (e.g., is DIP production a legitimate antiviral target?)?

How can we genetically modify viruses or manipulate host factors to inhibit or enhance DIP production? Can this knowledge be used to engineer virulent viruses into live attenuated vaccines by directing them to produce higher levels of DIPs?

How can we improve the sensitivity of assays to measure DIPs or their activity?

Many candidate DVGs are being identified using next-generation sequencing platforms. Are they able to mediate interference against standard infectious particles? While this question is difficult to answer, more direct testing is needed to move beyond correlative associations of a particular DIP/DVG signature and a particular disease state (i.e., enhanced or improved disease severity).

Highlights.

New technologies including reverse genetics, PCR, and next-generation sequencing have led to a resurgence in DIP research.

DVG synthesis was presumed to occur randomly due to the error-prone nature of viral polymerases. Recent work suggests that DVG generation can be a highly directed process.

Several novel viral and host factors required for DIP production have been discovered.

For certain viruses, DIP production appears to be a highly regulated and dynamic process. Importantly, it is now possible to engineer viruses that no longer create DIPs but still produce normal quantities of standard infectious virus particles.

DIPs have long been hypothesized to reduce virulence of a particular virus for its host. New evidence strongly suggests DIPs derived by several viruses including influenza and respiratory syncytial virus are protective in humans.

Glossary

Defective interfering particles (DIPs)

DIPs are biochemically similar to standard infectious virus particles, but differ because in most examples they package DVGs. DIPs are defective because they package a faulty genome and thus cannot complete the viral life cycle without help from standard infectious virus particles. DIPs are interfering because they block the propagation of standard infectious virus particles (in the setting where a host cell is co-infected with both a DIP and a standard infectious virus particle)

Defective viral genomes (DVGs)

viral genomes that arise through viral polymerase-driven mutation or recombination. They lack critical regions of the wild-type genome required for successful completion of the viral life cycle but retain the elements required for their replication by the viral polymerase. DVGs are the molecular basis for DIP-mediated interference with standard infectious virus particles. Specifically, DVGs outcompete wild-type genomes for access to viral genome replication machinery

Heterologous interference

a phenomenon whereby DIPs produced by a specific virus species can interfere with the propagation of standard infectious virus particles of a different virus species (i.e., LCMV DIPs interfering with Lassa virus standard virus particles)

Homologous interference

a phenomenon whereby DIPs produced by a specific virus species can interfere with the propagation of standard infectious virus particles produced by that same species of virus (i.e., LCMV DIPs interfering with LCMV standard virus particles)

Standard infectious virus particles

virus particles that package a wild-type viral genome. These virus particles can enter a host cell, replicate the viral genome, express the full complement of viral proteins, and successfully complete the viral life cycle by producing new infectious virions