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Defective interfering particles and virus evolution
R E V I E W S
36 Engstrand,L. et al. (1989) Infect. Immun. 57, 82%832 37 Crabtree,J.E. at al. (1991)Gut 32, 1473-1477 38 Crabtree,J.E. et el. (1993) Scand. J. Immunol. 37, 65-70 39 Fitzgerald,T.J. (1992) Infect. Immun. 60, 3475-3479 40 Karnes,W.E.J.et al. (1991) Gastroenterology 101,167-174 41 Cussac,V., Ferrero, R.L.and Labigne,A. (1992)J. Bacteriol. 174, 2466-2473 42 Frazier,B.A.et al. (1993)J. Bacteriol. 175, 966-972 43 Taylor,D.E. et al. (1992)J. Bacteriol. 174, 6800-6806 44 Akopyanz,N. et al. (1992) Nucleic Acids Res. 20, 5137-5142 45 Leunk,R.D. et al. (1988)J. Med. Microbiol. 26, 93-99 46 Cover,T.L., Dooley,C.P. and Blaser,M.J. (1990) Infect. Immun. 58,603-610 47 Figura,N. et al. (1989)J. Clin. Microbiol. 27, 225-226 48 Cover,T.L., Vaughn,S.G.and Blaser,M.J. (1992)]. Infect. Dis.
166, 1073-1078 49 Cover,T.L.et al. (1992)J. Clin. Invest. 90, 913-918 50 Cover,T.L.and Blaser,M.J. (1992)J. Biol. Chem. 267, 10570-10575 51 Cover,T.L., Reddy,L.Y.and Blaser,M.J. (1993) Infect. Immun. 61, 1427-1431 52 Apel,I. et al. (1988) Zentralbl. Bakteriol. Mikrobiol. Hyg. A 268, 271-276 53 Tummuru,M.K.R.,Cover,T.L.and Blaser,M.J. (1993) Infect. Immun. 61, 1799-1809 54 Crabtree,J.E. et el. (1991) Lancet 338, 332-335 55 Covacci,A. et al. Prec. Natl Acad. Sci. USA (in press) 56 Czinn,S., Cai, A. and Nedrud,J.G. (1992) Gastroenterology 102, A331 57 Clark, B.L.et al. (1974)Aust. Vet. J. 50, 407-409
Defective interfering particles and v i•r u s evolution Charles R.M. Bangham and Thomas B.L. Kirkwood efective interfering parAlmost all viruses produce replicationselection forces that act on Iticles (DIPs), discovered defective mutants that have complex DIPs and the force that DIPs by von Magnus in the effects on the growth and evolution of the themselves exert on viruses. early 1950s (Ref. 1), are natvirus in culture. These effects can be Other aspects of DIPs have urally occurring virus mutants explained qualitatively by a simple been reviewed recently s-v. In that lack part of the normal mathematical model. However, the model common with other hostviral genome. Because of this shows that the quantitative effects of these parasite interactions 8, an underthey are unable to replicate, mutants are intrinsically unpredictable. standing of the evolutionary unless the cell that they infect dynamics of DIPs can be gained C.I~.M. Bangham is in the Institute of Molecular is also infected with the norfrom theoretical models 9a°. Medicine and Dept of Virology, John Radcliffe mal parent or 'standard' virus, These studies show that DIP Hospital, Oxford, UK OX3 9DU; which contributes the genes replication shares important T.B.L. Kirkwood is in the National Institute that are missing or defective in characteristics with other hostfor Medical Research, Mill Hill, London, UK NW7 1AA. the DIP. The interesting effects parasite systems, such as a proof DIPs result from the fact nounced sensitivity to starting that they replicate at the expense of the standard virus conditions (i.e. the multiplicity of infection of DIP and in these co-infected cells; this is the phenomenon standard virus), and that one consequence of this is known as interference. Thus DIPs depend on stanthat the long-term outcome of infection is intrinsically dard virus to replicate and survive, but in replicating unpredictable. This has implications for the suggested they reduce the titre of standard virus. A good analtherapeutic uses of DIPs, and for the interpretation ogy can be drawn with the population dynamics of a of the effects of DIPs both in vitro and in v i v o . parasite (DIP) and its host (standard virus), with the further complication that both DIP and standard Effects of DIPs on virus growth virus depend on a supply of host cells. E f f e c t s in vitro DIPs are of practical importance for three main The fundamental effect of DIPs on virus replication reasons. First, they may modify natural virus infections. is to reduce the yield of infectious virus. The molSecond, it has been suggested that interfering viruses ecular mechanism is usually competition between the could be used therapeutically z. Third, they may serve DIP and standard virus genomes for replication and packaging 5,7. The term 'von Magnus effect' is used to as a tool to define the minimum viral sequences needed for replication and particle formation 3,4. describe the increase in the ratio of DIPs to standard In this review we summarize the effects of DIPs on virus that results from serial passage of the virus virus growth in vitro and in v i v o , and we examine the culture at high multiplicity of infection (m.o.i.), as role of DIPs in virus evolution, in terms of both the first described by von Magnus 1.
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Even in an acute infection, DIPs reduce the tempo of viral protein and polynucleotide synthesis 11. Indeed, lytic infection may be reduced to a level that allows continued growth of the cells, and a persistent infection is the result. In several instances, periodic fluctuations have been observed in the titre of standard virus and DIPs in persistent infections in vitro (Refs 7, 9 and references therein). Cave et al. 12 obtained evidence that fluctuating titres of vesicular stomatitis virus (VSV) in vivo were determined by DIP multiplicity. Occasionally, and apparently unpredictably, DIPs cause the extinction of infectious virus in vitroJ3; this has been called 'self-curing'. Mutants of standard virus commonly appear in vitro that are resistant to the interference effect of the DIP. These so-called Sdi- mutants have been described in many viruses 14-18, but the most extensively studied system has been VSV (Ref. 19 and references therein). DePolo et al. 19 followed a persistent VSV infection in vitro for over two years. They showed that from each Sdi- 'escape' mutant there arose, in turn, a new DIP to which it was susceptible. A new Sdi- mutant would then arise, and this succession seemed to continue indefinitely. The resistance of an Sdi- escape mutant to interference was usually restricted to just one DIP species, but the new DIP that arose to interfere with a given Sdi- mutant could generally interfere with the mutant's parent virus as well. Effects in vivo
Analysis of the actions of DIPs in animals is complicated by the wide variety of cell types infected, because different cells vary in their ability to form and propagate DIPs (see below), and because of the influence of an antigen-specific immune response. Also, DIPs are often efficient inducers of interferon production20, 21. DIPs can protect mice from a lethal infeGtion with VSV (Ref. 22), Semliki Forest virus 23 or influenza A virus 24. Cave et a l Y found a paradoxically higher protective effect of VSV DIPs at a lower input m.o.i. of DIPs, and they attributed this to the effect of a different DIP m.o.i, on the fluctuations in virus titre. That such paradoxical effects can be seen even in an acute infection emphasizes the need for caution when studying chronic infections, and still more if it is proposed to use DIPs therapeutically. Viruses with rearranged or deleted genomes have been isolated from many infected animals and humans 6, but in almost all cases it remains open to question whether these viruses behave as true DIPs in vivo. One clear example of attenuation of disease by DIPs was described by Chambers and Webster 2s, who showed that influenza A virus isolated from chickens attenuated influenza virus growth in experimentally inoculated birds in a dose-dependent way. The effects of DIPs in plants are more straightforward to analyse 7. DIPs of certain plant viruses undoubtedly attenuate the disease26,27; but in other cases DIPs can worsen disease 28.
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Mechanisms of DIP formation The mechanisms of DIP formation (and of their effects) have been reviewed recently 5-7. Two main types of molecular mechanism are believed to operate in DIP formation. "Jumping replicase" Most DIPs are formed when the viral replicase complex, carrying the nascent viral genome, falls off its template and reattaches at another site. If it rejoins a viral antigenome further downstream (Fig. la), the result is a virus genome with an internal deletion. If the nascent strand folds back on its own 5' end (Fig. lb), the result is a 'copy-back' or 'pan-handle' particle. Internally deleted DIPs are commoner, but some viruses, such as VSV, produce mainly copy-back DIPs. Interestingly, detachment of the replicase complex is an essential part of the replication of retroviruses, and deletion mutants of retroviruses are common 29. Certain artificial deletion mutants of retroviruses can dominantly interfere with replication, and this has given rise to speculation that such mutants of HIV might either cause or ameliorate AIDS (see Refs 2, 30 and references therein), although dominantly interfering mutants of retroviruses have not;been demonstrated in natural infections. Recombination
Genomic rearrangements are common during recombination, and duplication of replication or packaging signals may result in a DIP. Recombination is probably an important mechanism of DIP formation in DNA viruses; it is now clear that RNA viruses also frequently recombine 31.
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perimental usefulness of DIPs for making inferences about the minimum sequences involved in replication or packaging. While most DIPs do not need to make proteins, because the standard virus supplies the necessary genes, DIPs of both polioviruses44,4s and coronaviruses4°,46 frequently contain open reading frames, which can be strongly selected f o r 46. Presumably the proteins encoded by these open reading frames confer a selective advantage on the DIP, but the precise nature of this advantage has not yet been defined. Lastly, the host cell type can favour or even completely prevent DIP formation by a particular virus 6, while certain cell types can allow faithful propagation of DIPs but prevent their interfering effect. The reasons for this dependence on cell type are unknown, but it may account for the lack of a clear correlation between in vitro interference and in vivo protection by DIPs (Refs 6, 23).
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0.0001 0.001 0.01 0.1 1.0 Initial m.o.i, of standard virus (particles per cell) Fig. 2. Yield of standard virus (filled circles) and defective interfering particles (DIPs; open circles) in a single-passage culture. Here, 10 6 cells are infected and the culture is followed until the titres of standard and defective viruses in the supernatant reach a steady state. Each pair of curves is plotted at a constant initial ratio of standard virus to DIPs (1:1, solid line; 10:1, dashed line; 100:1, dotted line). At high initial standard virus m.o.i. (horizontal axis), the yield of standard virus is minimized and that of DIPs maximized. This illustrates the effect first observed by von Magnus': repeated passage of undiluted virus leads to the accumulation of DIPs. Redrawn from Ref. 9, with permission.
Selection constraints on the formation of DIPs
It is not certain to what extent the events that generate DIPs occur randomly over the virus genome, but several constraining factors have been identified that strongly affect the appearance and persistence of DIPs. The primary nucleotide sequence clearly influences the site of internal deletion in some cases. Nucleotide motifs either near 3z-34 o r a t 35,36 the deletion site have been identified, indicating sequence-directed template switching. Secondary RNA structure can determine the site of deletions introduced in polymerase chain reactions 37'38, but it has not been shown to influence DIP formation. The nucleotide sequence can also strongly determine the sites of point nucleotide substitutions introduced by RNA-dependent polymerases39. Point mutations are not a major mechanism of DIP generation, but they are important in the virus mutants that escape DIP interference (see above and Ref. 19). The small size of most DIP genomes gives them the selection advantage of more rapid replication than standard virus (Ref. 40 and references therein), although some DIPs are almost full-length4]. However, the efficiency of viral genome packaging decreases quickly as the size of the genome falls below a certain limit42,43. There is therefore an optimal size range for DIPs, above which they replicate slowly and below which they package inefficiently. This limits the ex-
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Virus evolution and DIPs
The increasing evidence of the extraordinary sequence variability of viruses, particularly RNA viruses47,48 makes it clear that the rate-limiting factors in the evolution of a virus may lie less in its mutation or recombination rate and more in the selection forces that act on it, either to constrain variation (to preserve function) or to favour variation (for example to escape immune recognition) 49. There are two aspects of virus evolution to consider. The first concerns the longterm evolution of the virus in the population (i.e. sequence polymorphism or fixation of mutations), while the second concerns the shorter-term changes that arise during the course of an individual infection. Here we are concerned mainly with the short-term selection pressure exerted by DIPs. Clearly there is an interplay between the shortterm changes in the virus that occur during individual infections and the evolution of the virus in the population, but DIPs are unlikely to contribute significantly to the latter process, at least in animals. First, virus escape from interference is usually specific to a particular DIP. Second, the production and effects of DIPs depend strongly on host cell type and may vary from tissue to tissue. For these two reasons it is hard to imagine an interference escape mutant that has a general survival advantage in the population. Third, in the process of transmission of virus from one animal host to another, any selection pressure exerted by DIPs will be relaxed and may be absent altogether. Most naturally acquired infections begin as a low m.o.i, infection of host cells, and the infection is short-lived as it is limited by the immune response. These conditions favour the growth of standard virus rather than DIPs. Thus, in the transmission of virus infections the dominant selection forces are likely to be related to factors such as infectivity, tissue tropism and speed of virus replication. These are determined primarily by the relationship of the standard virus to its host. In plants, none of these constraints is so important: there is no antigen-specific adaptive immune system,
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there are far fewer differentiated cell types, and the virus inoculum may be high because of the quantity of parental material transferred during vegetative propagation. It is therefore easier to conceive that DIPs will influence the evolution of plant viruses at the population level. A model of DIP growth
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Because the effects of DIPs, even in 'N, , , 0J well-controlled experiments, are often I ! complex and unpredictable, it is frequently suggested that stochastic DIP (chance) events strongly influence 7 r the propagation of DIPs, or that D1 D2 D3 D4 other factors (such as interferon, 6 / I ¢ l . temperature-sensitive effects or mus . I ~/ v I / I I v I I / ~# i ! I ~ I Y I / I/ II tations in the ho~t cell) modify the ~" /, V ',,' ,/ /, course of an infection. While we ~1 ' V I; were in no doubt that each of these j '~ factors can modify an individual in- / fection, we wanted to test whether " the complex effects of DIPs on virus ! growth could be explained more 0 , I I I I I I I simply, using only what is already 0 10 20 30' 40 50 60 70 known about their effects at the Passage single cell level. We made the following assumpFig. 3. A simulation of how defective interfering particles (DIPs) may drive the evolution of tions9: (1) cells infected only with virus in vitro. The initial standard virus $1 (upper panel) produces a DIP, D1 (lower panel), DIPs grow normally, (2) standard within one passage (mutation rate = 10~). When a new standard virus, $2, emerges that escapes the interfering effect of D1, first D1 and then $1 is extinguished. This effect begins virus and DIPs infect cells at the only when the log titre of $2 reaches about 4. $2 in turn produces a new DIP, D2, and this same rate per cell per virion (we pattern of replacement continues. Note that (1) the survival times of the viral strains vary assume perfect mixing), (3) coconsiderably, and (2) not all standard virus escape mutants become established (arrows, infected cells produce only DIPs upper panel). (although less strong interference produces qualitatively similar results) and (4) the DIP burst size (number of DIPs produced model simulates the kind of quasiperiodic fluctuation per cell) is equal to the standard virus burst size that is seen in experiments, but that is unpredictable (again, this can be varied without qualitatively in its fine detail. changing the outcome). The numerical values of virus Unexpectedly, the model also accounts for the growth rate etc. were derived from experimental 'self-curing' effect of DIPs, i.e. the loss of infectious data, and the model was simulated deterministically virus in an in vitro culture initially containing both on a computer. infectious virus and DIPs. This behaviour is explained A requirement of the model was that it should as follows. When the initial DIP m.o.i, is very high, explain the von Magnus effect. That it does so is most cells are either Coinfected or infected with DIPs shown in Fig. 2" whatever the input ratio of standard only, so that only DIPs are produced. As soon as any virus to DIPs, increasing the concentration of the coinfected cells have released virus and died, only inoculum (i.e. following a pair of curves to the right) cells carrying DIPs alone are left, and these grow nordecreases the standard:DIP ratio after one passage. mally while absorbing the remaining DIPs from the Thus, repeated passage of a highly concentrated culture medium. Only when the initial m.o.i, of the inoculum leads to the accumulation of DIPs. standard virus is equally high is the more expected Simulation of repeated passage of a diluted inoculum outcome observed, in this case because sufficient cells readily produces the cyclical variation in virus titres were infected with standard virus alone in the initial that has been observed (see above). It is important to round of infections. The prediction by the model that note that, as seen in experiments in vitro, the cycling DIPs remain in the cell monolayer after the eradiis periodic, but not completely regular. This pattern cation of replicating virus is remarkably similar to the of variation is strongly reminiscent of the mathematfinding of Jacobson et aI.13 that viral antigens can be ical phenomenon of 'deterministic chaos', in which identified by immunofluorescence after the apparent reiteration of a simple deterministic equation leads to self-curing of the culture. This unanticipated result results that are in principle unpredictable, or 'chaotic', increases our confidence that the model is soundly beyond a short time. Thus a wholly deterministic based.
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Recently, we have extended the model to examine the selection effects of DIPs on virus evolution in vitro (T.B.L. Kirkwood and C.R.M. Bangham, unpublished). The viruses are now permitted to mutate randomly at a given rate, to produce either a new 'escape' mutant that is immune to interference by the current DIP, or a new DIP that can interfere with both the current 'escape' mutant and its parent virus. An example of a simulated experiment is shown in Fig. 3. It is striking that the model predicts a succession of new escape mutants and new DIPs, similar to those observed experimentally 19. The driving force in this process is the selection pressure exerted by interference. Because the success or failure of a new escape mutant is strongly dependent on the frequencies of standard virus and DIPs at the time it first appears in the culture, and because of the irregular fluctuations in virus titre already noted, we see that the lifetimes of individual variants in the simulated experiment can vary widely, in an intrinsically unpredictable way. The model is proving useful to explore the conditions favouring the establishment and survival of new mutations within the cultures. Conclusions
There are two conclusions from this work. First, the complex effects of DIPs on the population dynamics of viruses in vitro can be accounted for qualitatively using only what is known about the actions of DIPs on a single infected cell. Second, the complex dynamics observed, which strongly suggest deterministic chaos, make it clear that even under idealized conditions in vitro, the outcome of an infection in q u a n t i t a t i v e terms is logically impossible to predict after only a short time. Also, it is impossible to infer events at the cellular level from the pattern of variation seen at the population level, and it is not necessary to invoke such events to explain the unpredictable features of DIP growth. There is also an implication for the suggested use of DIPs, or the analogous dominantly interfering virus mutants, in the treatment of diseases such as AIDS (Ref. 2). The quantitative effects of such viruses in an infection in vivo, particularly in a chronic infection, will be highly unpredictable, and this suggests that such agents should only be used with great care. References
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36 Engstrand,L. et al. (1989) Infect. Immun. 57, 82%832 37 Crabtree,J.E. at al. (1991)Gut 32, 1473-1477 38 Crabtree,J.E. et el. (1993) Scand. J. Immunol. 37, 65-70 39 Fitzgerald,T.J. (1992) Infect. Immun. 60, 3475-3479 40 Karnes,W.E.J.et al. (1991) Gastroenterology 101,167-174 41 Cussac,V., Ferrero, R.L.and Labigne,A. (1992)J. Bacteriol. 174, 2466-2473 42 Frazier,B.A.et al. (1993)J. Bacteriol. 175, 966-972 43 Taylor,D.E. et al. (1992)J. Bacteriol. 174, 6800-6806 44 Akopyanz,N. et al. (1992) Nucleic Acids Res. 20, 5137-5142 45 Leunk,R.D. et al. (1988)J. Med. Microbiol. 26, 93-99 46 Cover,T.L., Dooley,C.P. and Blaser,M.J. (1990) Infect. Immun. 58,603-610 47 Figura,N. et al. (1989)J. Clin. Microbiol. 27, 225-226 48 Cover,T.L., Vaughn,S.G.and Blaser,M.J. (1992)]. Infect. Dis.
166, 1073-1078 49 Cover,T.L.et al. (1992)J. Clin. Invest. 90, 913-918 50 Cover,T.L.and Blaser,M.J. (1992)J. Biol. Chem. 267, 10570-10575 51 Cover,T.L., Reddy,L.Y.and Blaser,M.J. (1993) Infect. Immun. 61, 1427-1431 52 Apel,I. et al. (1988) Zentralbl. Bakteriol. Mikrobiol. Hyg. A 268, 271-276 53 Tummuru,M.K.R.,Cover,T.L.and Blaser,M.J. (1993) Infect. Immun. 61, 1799-1809 54 Crabtree,J.E. et el. (1991) Lancet 338, 332-335 55 Covacci,A. et al. Prec. Natl Acad. Sci. USA (in press) 56 Czinn,S., Cai, A. and Nedrud,J.G. (1992) Gastroenterology 102, A331 57 Clark, B.L.et al. (1974)Aust. Vet. J. 50, 407-409
Defective interfering particles and v i•r u s evolution Charles R.M. Bangham and Thomas B.L. Kirkwood efective interfering parAlmost all viruses produce replicationselection forces that act on Iticles (DIPs), discovered defective mutants that have complex DIPs and the force that DIPs by von Magnus in the effects on the growth and evolution of the themselves exert on viruses. early 1950s (Ref. 1), are natvirus in culture. These effects can be Other aspects of DIPs have urally occurring virus mutants explained qualitatively by a simple been reviewed recently s-v. In that lack part of the normal mathematical model. However, the model common with other hostviral genome. Because of this shows that the quantitative effects of these parasite interactions 8, an underthey are unable to replicate, mutants are intrinsically unpredictable. standing of the evolutionary unless the cell that they infect dynamics of DIPs can be gained C.I~.M. Bangham is in the Institute of Molecular is also infected with the norfrom theoretical models 9a°. Medicine and Dept of Virology, John Radcliffe mal parent or 'standard' virus, These studies show that DIP Hospital, Oxford, UK OX3 9DU; which contributes the genes replication shares important T.B.L. Kirkwood is in the National Institute that are missing or defective in characteristics with other hostfor Medical Research, Mill Hill, London, UK NW7 1AA. the DIP. The interesting effects parasite systems, such as a proof DIPs result from the fact nounced sensitivity to starting that they replicate at the expense of the standard virus conditions (i.e. the multiplicity of infection of DIP and in these co-infected cells; this is the phenomenon standard virus), and that one consequence of this is known as interference. Thus DIPs depend on stanthat the long-term outcome of infection is intrinsically dard virus to replicate and survive, but in replicating unpredictable. This has implications for the suggested they reduce the titre of standard virus. A good analtherapeutic uses of DIPs, and for the interpretation ogy can be drawn with the population dynamics of a of the effects of DIPs both in vitro and in v i v o . parasite (DIP) and its host (standard virus), with the further complication that both DIP and standard Effects of DIPs on virus growth virus depend on a supply of host cells. E f f e c t s in vitro DIPs are of practical importance for three main The fundamental effect of DIPs on virus replication reasons. First, they may modify natural virus infections. is to reduce the yield of infectious virus. The molSecond, it has been suggested that interfering viruses ecular mechanism is usually competition between the could be used therapeutically z. Third, they may serve DIP and standard virus genomes for replication and packaging 5,7. The term 'von Magnus effect' is used to as a tool to define the minimum viral sequences needed for replication and particle formation 3,4. describe the increase in the ratio of DIPs to standard In this review we summarize the effects of DIPs on virus that results from serial passage of the virus virus growth in vitro and in v i v o , and we examine the culture at high multiplicity of infection (m.o.i.), as role of DIPs in virus evolution, in terms of both the first described by von Magnus 1.
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Even in an acute infection, DIPs reduce the tempo of viral protein and polynucleotide synthesis 11. Indeed, lytic infection may be reduced to a level that allows continued growth of the cells, and a persistent infection is the result. In several instances, periodic fluctuations have been observed in the titre of standard virus and DIPs in persistent infections in vitro (Refs 7, 9 and references therein). Cave et al. 12 obtained evidence that fluctuating titres of vesicular stomatitis virus (VSV) in vivo were determined by DIP multiplicity. Occasionally, and apparently unpredictably, DIPs cause the extinction of infectious virus in vitroJ3; this has been called 'self-curing'. Mutants of standard virus commonly appear in vitro that are resistant to the interference effect of the DIP. These so-called Sdi- mutants have been described in many viruses 14-18, but the most extensively studied system has been VSV (Ref. 19 and references therein). DePolo et al. 19 followed a persistent VSV infection in vitro for over two years. They showed that from each Sdi- 'escape' mutant there arose, in turn, a new DIP to which it was susceptible. A new Sdi- mutant would then arise, and this succession seemed to continue indefinitely. The resistance of an Sdi- escape mutant to interference was usually restricted to just one DIP species, but the new DIP that arose to interfere with a given Sdi- mutant could generally interfere with the mutant's parent virus as well. Effects in vivo
Analysis of the actions of DIPs in animals is complicated by the wide variety of cell types infected, because different cells vary in their ability to form and propagate DIPs (see below), and because of the influence of an antigen-specific immune response. Also, DIPs are often efficient inducers of interferon production20, 21. DIPs can protect mice from a lethal infeGtion with VSV (Ref. 22), Semliki Forest virus 23 or influenza A virus 24. Cave et a l Y found a paradoxically higher protective effect of VSV DIPs at a lower input m.o.i. of DIPs, and they attributed this to the effect of a different DIP m.o.i, on the fluctuations in virus titre. That such paradoxical effects can be seen even in an acute infection emphasizes the need for caution when studying chronic infections, and still more if it is proposed to use DIPs therapeutically. Viruses with rearranged or deleted genomes have been isolated from many infected animals and humans 6, but in almost all cases it remains open to question whether these viruses behave as true DIPs in vivo. One clear example of attenuation of disease by DIPs was described by Chambers and Webster 2s, who showed that influenza A virus isolated from chickens attenuated influenza virus growth in experimentally inoculated birds in a dose-dependent way. The effects of DIPs in plants are more straightforward to analyse 7. DIPs of certain plant viruses undoubtedly attenuate the disease26,27; but in other cases DIPs can worsen disease 28.
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Mechanisms of DIP formation The mechanisms of DIP formation (and of their effects) have been reviewed recently 5-7. Two main types of molecular mechanism are believed to operate in DIP formation. "Jumping replicase" Most DIPs are formed when the viral replicase complex, carrying the nascent viral genome, falls off its template and reattaches at another site. If it rejoins a viral antigenome further downstream (Fig. la), the result is a virus genome with an internal deletion. If the nascent strand folds back on its own 5' end (Fig. lb), the result is a 'copy-back' or 'pan-handle' particle. Internally deleted DIPs are commoner, but some viruses, such as VSV, produce mainly copy-back DIPs. Interestingly, detachment of the replicase complex is an essential part of the replication of retroviruses, and deletion mutants of retroviruses are common 29. Certain artificial deletion mutants of retroviruses can dominantly interfere with replication, and this has given rise to speculation that such mutants of HIV might either cause or ameliorate AIDS (see Refs 2, 30 and references therein), although dominantly interfering mutants of retroviruses have not;been demonstrated in natural infections. Recombination
Genomic rearrangements are common during recombination, and duplication of replication or packaging signals may result in a DIP. Recombination is probably an important mechanism of DIP formation in DNA viruses; it is now clear that RNA viruses also frequently recombine 31.
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perimental usefulness of DIPs for making inferences about the minimum sequences involved in replication or packaging. While most DIPs do not need to make proteins, because the standard virus supplies the necessary genes, DIPs of both polioviruses44,4s and coronaviruses4°,46 frequently contain open reading frames, which can be strongly selected f o r 46. Presumably the proteins encoded by these open reading frames confer a selective advantage on the DIP, but the precise nature of this advantage has not yet been defined. Lastly, the host cell type can favour or even completely prevent DIP formation by a particular virus 6, while certain cell types can allow faithful propagation of DIPs but prevent their interfering effect. The reasons for this dependence on cell type are unknown, but it may account for the lack of a clear correlation between in vitro interference and in vivo protection by DIPs (Refs 6, 23).
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0.0001 0.001 0.01 0.1 1.0 Initial m.o.i, of standard virus (particles per cell) Fig. 2. Yield of standard virus (filled circles) and defective interfering particles (DIPs; open circles) in a single-passage culture. Here, 10 6 cells are infected and the culture is followed until the titres of standard and defective viruses in the supernatant reach a steady state. Each pair of curves is plotted at a constant initial ratio of standard virus to DIPs (1:1, solid line; 10:1, dashed line; 100:1, dotted line). At high initial standard virus m.o.i. (horizontal axis), the yield of standard virus is minimized and that of DIPs maximized. This illustrates the effect first observed by von Magnus': repeated passage of undiluted virus leads to the accumulation of DIPs. Redrawn from Ref. 9, with permission.
Selection constraints on the formation of DIPs
It is not certain to what extent the events that generate DIPs occur randomly over the virus genome, but several constraining factors have been identified that strongly affect the appearance and persistence of DIPs. The primary nucleotide sequence clearly influences the site of internal deletion in some cases. Nucleotide motifs either near 3z-34 o r a t 35,36 the deletion site have been identified, indicating sequence-directed template switching. Secondary RNA structure can determine the site of deletions introduced in polymerase chain reactions 37'38, but it has not been shown to influence DIP formation. The nucleotide sequence can also strongly determine the sites of point nucleotide substitutions introduced by RNA-dependent polymerases39. Point mutations are not a major mechanism of DIP generation, but they are important in the virus mutants that escape DIP interference (see above and Ref. 19). The small size of most DIP genomes gives them the selection advantage of more rapid replication than standard virus (Ref. 40 and references therein), although some DIPs are almost full-length4]. However, the efficiency of viral genome packaging decreases quickly as the size of the genome falls below a certain limit42,43. There is therefore an optimal size range for DIPs, above which they replicate slowly and below which they package inefficiently. This limits the ex-
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Virus evolution and DIPs
The increasing evidence of the extraordinary sequence variability of viruses, particularly RNA viruses47,48 makes it clear that the rate-limiting factors in the evolution of a virus may lie less in its mutation or recombination rate and more in the selection forces that act on it, either to constrain variation (to preserve function) or to favour variation (for example to escape immune recognition) 49. There are two aspects of virus evolution to consider. The first concerns the longterm evolution of the virus in the population (i.e. sequence polymorphism or fixation of mutations), while the second concerns the shorter-term changes that arise during the course of an individual infection. Here we are concerned mainly with the short-term selection pressure exerted by DIPs. Clearly there is an interplay between the shortterm changes in the virus that occur during individual infections and the evolution of the virus in the population, but DIPs are unlikely to contribute significantly to the latter process, at least in animals. First, virus escape from interference is usually specific to a particular DIP. Second, the production and effects of DIPs depend strongly on host cell type and may vary from tissue to tissue. For these two reasons it is hard to imagine an interference escape mutant that has a general survival advantage in the population. Third, in the process of transmission of virus from one animal host to another, any selection pressure exerted by DIPs will be relaxed and may be absent altogether. Most naturally acquired infections begin as a low m.o.i, infection of host cells, and the infection is short-lived as it is limited by the immune response. These conditions favour the growth of standard virus rather than DIPs. Thus, in the transmission of virus infections the dominant selection forces are likely to be related to factors such as infectivity, tissue tropism and speed of virus replication. These are determined primarily by the relationship of the standard virus to its host. In plants, none of these constraints is so important: there is no antigen-specific adaptive immune system,
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there are far fewer differentiated cell types, and the virus inoculum may be high because of the quantity of parental material transferred during vegetative propagation. It is therefore easier to conceive that DIPs will influence the evolution of plant viruses at the population level. A model of DIP growth
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Because the effects of DIPs, even in 'N, , , 0J well-controlled experiments, are often I ! complex and unpredictable, it is frequently suggested that stochastic DIP (chance) events strongly influence 7 r the propagation of DIPs, or that D1 D2 D3 D4 other factors (such as interferon, 6 / I ¢ l . temperature-sensitive effects or mus . I ~/ v I / I I v I I / ~# i ! I ~ I Y I / I/ II tations in the ho~t cell) modify the ~" /, V ',,' ,/ /, course of an infection. While we ~1 ' V I; were in no doubt that each of these j '~ factors can modify an individual in- / fection, we wanted to test whether " the complex effects of DIPs on virus ! growth could be explained more 0 , I I I I I I I simply, using only what is already 0 10 20 30' 40 50 60 70 known about their effects at the Passage single cell level. We made the following assumpFig. 3. A simulation of how defective interfering particles (DIPs) may drive the evolution of tions9: (1) cells infected only with virus in vitro. The initial standard virus $1 (upper panel) produces a DIP, D1 (lower panel), DIPs grow normally, (2) standard within one passage (mutation rate = 10~). When a new standard virus, $2, emerges that escapes the interfering effect of D1, first D1 and then $1 is extinguished. This effect begins virus and DIPs infect cells at the only when the log titre of $2 reaches about 4. $2 in turn produces a new DIP, D2, and this same rate per cell per virion (we pattern of replacement continues. Note that (1) the survival times of the viral strains vary assume perfect mixing), (3) coconsiderably, and (2) not all standard virus escape mutants become established (arrows, infected cells produce only DIPs upper panel). (although less strong interference produces qualitatively similar results) and (4) the DIP burst size (number of DIPs produced model simulates the kind of quasiperiodic fluctuation per cell) is equal to the standard virus burst size that is seen in experiments, but that is unpredictable (again, this can be varied without qualitatively in its fine detail. changing the outcome). The numerical values of virus Unexpectedly, the model also accounts for the growth rate etc. were derived from experimental 'self-curing' effect of DIPs, i.e. the loss of infectious data, and the model was simulated deterministically virus in an in vitro culture initially containing both on a computer. infectious virus and DIPs. This behaviour is explained A requirement of the model was that it should as follows. When the initial DIP m.o.i, is very high, explain the von Magnus effect. That it does so is most cells are either Coinfected or infected with DIPs shown in Fig. 2" whatever the input ratio of standard only, so that only DIPs are produced. As soon as any virus to DIPs, increasing the concentration of the coinfected cells have released virus and died, only inoculum (i.e. following a pair of curves to the right) cells carrying DIPs alone are left, and these grow nordecreases the standard:DIP ratio after one passage. mally while absorbing the remaining DIPs from the Thus, repeated passage of a highly concentrated culture medium. Only when the initial m.o.i, of the inoculum leads to the accumulation of DIPs. standard virus is equally high is the more expected Simulation of repeated passage of a diluted inoculum outcome observed, in this case because sufficient cells readily produces the cyclical variation in virus titres were infected with standard virus alone in the initial that has been observed (see above). It is important to round of infections. The prediction by the model that note that, as seen in experiments in vitro, the cycling DIPs remain in the cell monolayer after the eradiis periodic, but not completely regular. This pattern cation of replicating virus is remarkably similar to the of variation is strongly reminiscent of the mathematfinding of Jacobson et aI.13 that viral antigens can be ical phenomenon of 'deterministic chaos', in which identified by immunofluorescence after the apparent reiteration of a simple deterministic equation leads to self-curing of the culture. This unanticipated result results that are in principle unpredictable, or 'chaotic', increases our confidence that the model is soundly beyond a short time. Thus a wholly deterministic based.
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Recently, we have extended the model to examine the selection effects of DIPs on virus evolution in vitro (T.B.L. Kirkwood and C.R.M. Bangham, unpublished). The viruses are now permitted to mutate randomly at a given rate, to produce either a new 'escape' mutant that is immune to interference by the current DIP, or a new DIP that can interfere with both the current 'escape' mutant and its parent virus. An example of a simulated experiment is shown in Fig. 3. It is striking that the model predicts a succession of new escape mutants and new DIPs, similar to those observed experimentally 19. The driving force in this process is the selection pressure exerted by interference. Because the success or failure of a new escape mutant is strongly dependent on the frequencies of standard virus and DIPs at the time it first appears in the culture, and because of the irregular fluctuations in virus titre already noted, we see that the lifetimes of individual variants in the simulated experiment can vary widely, in an intrinsically unpredictable way. The model is proving useful to explore the conditions favouring the establishment and survival of new mutations within the cultures. Conclusions
There are two conclusions from this work. First, the complex effects of DIPs on the population dynamics of viruses in vitro can be accounted for qualitatively using only what is known about the actions of DIPs on a single infected cell. Second, the complex dynamics observed, which strongly suggest deterministic chaos, make it clear that even under idealized conditions in vitro, the outcome of an infection in q u a n t i t a t i v e terms is logically impossible to predict after only a short time. Also, it is impossible to infer events at the cellular level from the pattern of variation seen at the population level, and it is not necessary to invoke such events to explain the unpredictable features of DIP growth. There is also an implication for the suggested use of DIPs, or the analogous dominantly interfering virus mutants, in the treatment of diseases such as AIDS (Ref. 2). The quantitative effects of such viruses in an infection in vivo, particularly in a chronic infection, will be highly unpredictable, and this suggests that such agents should only be used with great care. References
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