©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
What Reassorts When Reovirus Genome Segments Reassort? (*)

Wolfgang K. Joklik Michael R. Roner

From the Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710

INTRODUCTION
The Packaging of the Three Single-stranded 6 Genome Segment Precursors
The Nature of Reovirus RNA Assortment Complexes
The Infectious Reovirus RNA System
The Nature of the Genome Segments in Reovirus Reassortants
What Is the Significance of Mutations in Reovirus Reassortants?
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

The members of several families of RNA-containing viruses possess segmented genomes that consist of from 2 to 12 genome segments. These viruses have the problem of assembling their genome segments into sets that contain one of each, a process that is usually referred to as assortment. Viruses appear to solve this problem in two ways, exemplified by influenza virus, on the one hand, and by reovirus, on the other. Influenza virus possesses eight species of negative stranded genome segments, each tightly associated with the nucleocapsid protein NP(1) . Since these segments are presumably unable to identify and recognize each other, it has been proposed that there is in fact no specific assortment mechanism for the influenza virus genome but that influenza virus particles contain more than 8 (say 11) randomly assorted genome segments so that 1 in about every 20-30 virus particles, on average, will contain one of each of the eight genome segments and, therefore, be viable(2, 3) .

Reovirus, on the other hand, must use a specific assortment mechanism because here the ratio of total to infectious virus particles is less than 2(4, 5) . The genome of reovirus consists of 10 species of double-stranded RNA molecules; and reovirologists have long been fascinated by the possible nature of an assortment mechanism that assembles 10 unique species of RNA molecules with very high specificity and efficiency. The key observation here was the demonstration of genome segment reassortment, that is the virtually unrestricted incorporation of genome segments from two parental virus particles into single genomes, as demonstrated by the fact that about 15% of the progeny arising in cells simultaneously infected with two ts (^1)mutants are ts virus (6) . This phenomenon of genome segment reassortment was analyzed in detail in studies of the progeny of pairs of the three reovirus serotypes, the genomes of each of which are homologous genome segment sets(7, 8) . This analysis demonstrated virtually random, that is unlinked, genome segment reassortment(9, 10, 11) . These studies led to four assumptions that were never tested. The first is that the genome segments being reassorted were the same as the genome segments being assorted, that is, the vast majority of wt population components. There was no reason for this assumption to be suspect; after all, even if the error frequency at each locus were as high as 10, then even among 4000-residue-long L-size class reovirus genome segments more than 95% would be wt. Second, there has never been serious consideration of whether the transcripts of parental genomes that function as messenger RNAs are the same as those that are assorted into genomes; it is conceivable that some modification occurs that marks transcripts for either one function or the other. Third, it is assumed that the genome segments that are assorted into genomes are components of a pool from which they are withdrawn at random. It is, however, conceivable that the distribution of ss transcripts synthesized by parental particles is compartmentalized; for example, some transcripts may be formed within a scaffolding mechanism from which they are never liberated and within which progeny genomes are assembled. The fourth assumption is that the molecules being selected for incorporation into genomes are ss rather than dsRNA molecules (12) (since the latter would be unable to interact via complementary sequences).

Recent evidence suggests that there may be problems with each of these assumptions.

The fact that reassortment is more complicated than pools of ssRNA genome segments transcribed from two parental genomes interacting randomly was suggested several years ago when parental genomes were introduced into cells not as infectious virus particles but as microinjected cores (13) (the specific infectivity of which is the same as that of virus particles, their only handicap being inability to attach to cells via interaction of the cell attachment protein 1 (14) (which they lack) with receptors). In the genomes of the reassortant populations formed in cells infected with the virions of any two of the three serotypes of reovirus, the distribution of genome segments of the two parents is more or less Poissonian, that is, there is little linkage between any segments(9) . In sharp contrast, the reassortants that result from infection with cores of the two parents still account for about 15% of yields, but they are now almost exclusively monoreassortants (MRs), that is they contain 9 genome segments of one parent and 1 from another(13) . And when single ss genome segments of one serotype are injected into cells infected with virions of another, these single segments are not incorporated into viral genomes. Thus reovirus genome segment assortment/reassortment is clearly a more complex process than simply selecting genome segments from pools (Fig. 1).


Figure 1: The reovirus multiplication cycle.



Before we examine in some detail the insight gained from recent studies of the mechanisms involved in reovirus genome segment assortment/reassortment, it is instructive to review work on dsRNA genome segment assortment and packaging in a prokaryotic virus system.


The Packaging of the Three Single-stranded 6 Genome Segment Precursors

The genome of bacteriophage 6 consists of three segments of dsRNA, the largest of which encodes four proteins, P1, P2, P4, and P7, which self-assemble into dodecahedral structures known as procapsids (15, 16, 17) . These procapsids are capable of packaging, in vitro, the three species of 6 ssRNA in a manner that suggests that each procapsid contains one specific binding site for each (18, 19, 20) , but the nature of these binding sites is unknown. Since the presence of P2, the 6 RNA polymerase, is necessary for binding to occur, it is assumed that P2 is a component of the binding sites(19) , but whether all three sites are on one, or on three, molecules of P2 and whether and how any of the other procapsid proteins are also involved in binding is not known. Nor is it known how only one binding site can be generated in highly symmetrical particles like procapsids, each of which contains multiple copies of each of its four component protein species, including P2 (of which there are 10-20 molecules present in each procapsid). The favored current hypothesis is that in the bacterial cell a single P2 molecule with three binding sites nucleates formation of procapsids(21) . Interestingly, transcription of the three plus strands into minus strands requires the presence of all three 5` termini, but not of the three 3` termini, which suggests that occupation of all three binding sites triggers some conformational change in P2 (which, again, could be more easily imagined if only one, rather than three, P2 molecules were involved)(20) .

To summarize, there are three separate binding sites for the three species of 6 ssRNA, which do not have to interact or recognize each other; the binding sites are on the RNA polymerase; and the process of RNA packaging is separate from the synthesis of minus strand RNA.


The Nature of Reovirus RNA Assortment Complexes

There is a long history of attempts to discover the nature of the complexes within which reovirus genome segments are assorted. These attempts took the form of physically isolating, either by density gradient centrifugation or by gel electrophoresis under nondenaturing conditions, complexes or particles with the ability to transcribe endogenous plus strands into minus strands and their immediate precursors. ``Fractions'' containing such complexes (``replicase particles'') were readily obtained(22, 23, 24, 25, 26, 27, 28) . Although isolating them proved to be impossible, rough estimates could be obtained of their size and protein composition. ``Replicase particles'' are about 40 nm in diameter; their sedimentation coefficients range from 180 to 550 S; their plus-stranded RNA templates are sensitive to RNase digestion; and their major protein components are the core components 1, 2, and 3, together with µ1C, 2, and 3. Interestingly, the particles with the lowest sedimentation coefficients synthesized predominantly genome segments of the S size class; those with intermediate S values synthesized increased amounts of M size class genome segments; and those with the largest S values synthesized predominantly L size class genome segments. There was no synthesis of plus strands in these particles, nor was there initiation (only completion) of minus strand synthesis; and once synthesis of minus strands was complete, all particles sedimented with about 550 S. It was not demonstrated, however, that each of these particle species contained one, and only one, of each species of plus-stranded RNA template strands, that is that they contained already assorted genome segment sets.

More recently similar studies have been carried out with rotaviruses (29, 30, 31) . Here three species of ``replicase particles'' were identified. First, there are the ``pre-core RIs'' (replicative intermediates) (45 nm, 220 S), which contain the core proteins VP1 (the polymerase) and VP3 (the guanylyltransferase), as well as VP9 and the nonstructural RNA-binding proteins NS53, NS35, and NS34. These particles do not yet possess RNA polymerase activity. Then there are the ``core RIs'' (60 nm, 310 S), which contain, in addition, the major core component VP2 (but have lost NS53) and which do possess polymerase activity. Finally there are the largest RIs, which contain, in addition, the outer single-shell particle component VP6 (75 nm, 420 S). When these particles were incubated in a cell-free system capable of supporting RNA synthesis, they underwent continuous size changes caused by the recruitment of plus strands; and synthesis of full-length dsRNAs proceeded, with time, from the smallest to the largest genome segment.

Since it is difficult, if not impossible, to isolate assortment complexes in pure form, attempts have recently been made to identify, as the virus multiplication cycle proceeds, which reovirus proteins are associated, first with viral ssRNA and then with viral dsRNA, using as the criterion of association the ability of highly specific antibodies against reovirus proteins to precipitate viral RNA(32) . It turns out that plus-stranded reovirus RNA molecules (that is the ss forms of genome segments) associate with three reovirus proteins within minutes after they are transcribed: the two nonstructural proteins µNS and NS, and protein 3(32) . The fact that NS is an ssRNA-binding protein has been known for some time(33) . While this binding is not sequence-specific, it is very strong; in fact, it is difficult to remove final traces of RNA during NS purification. Protein µNS, on the other hand, had not previously been known to be an RNA-binding protein; yet in infected cells it is the first reovirus protein to bind to ssRNA, that is, its binding of ssRNA is not dependent on some other reovirus-specified protein also binding. As for 3, it binds in vitro to ds, not to ssRNA(33) ; it is therefore suspected that what is bound in vivo is some ds feature of reovirus ss genome segments such as hairpin loops or intra- or interstrand complementary sequences; such binding most probably is structure- or shape-specific, rather than sequence-specific.

Each of these ssRNA-containing complexes (ssRCCs) contains one ssRNA molecule and, depending on its size (reovirus genome segments are from 1200 to 3900 bp long), 10-30 protein molecules. Almost all ssRCCs contain µNS; about one-half contain µNS alone; about one-half contain 3; and about one-quarter contain NS. As for the relative proportions of the individual RNA species in ssRCCs, these reflect the composition of the total ssRNA population present in infected cells, which differs substantially from equimolarity(34) .

Four hours after infection, complexes appear that contain dsRNA rather than ssRNA (dsRCCs); and these complexes contain not only the same three proteins as are also present in ssRCCs but also 2 (and, presumably, the reovirus RNA polymerase, the catalytic component of which is protein 3). The highly significant feature of dsRCCs is that the relative proportions of the 10 dsRNA species in them are equimolar. This suggests that genome segment assortment into progeny genomes is linked to the transcription of plus strands into minus strands, and that the factor that controls assortment is, again, the RNA polymerase.


The Infectious Reovirus RNA System

Another approach to identifying the signals required to assort reovirus genome segments into genomes involves the recently elaborated infectious reovirus RNA system(35) . The significance of this system is as follows. After conversion of infecting parental virions into subviral particles, the dsRNA genome segments within them are transcribed into plus strands by enzymes (polymerase and capping enzymes) present within them. Since these 10 ssRNA transcripts are the only genetic/biochemical link between parental and progeny genomes, infectious reovirus particles should be formed in cells into which all 10 species of ssRNA molecules are introduced; and conditions have recently been found under which reovirus RNA is indeed infectious(35) . This system has the potential for introducing novel genetic information into the reovirus genome; not only should it permit identification of the sequences on genome segments that function as assortment/reassortment signals, but also identification of the functional domains of the various reovirus-specified proteins. The latter should, in turn, permit the study of the effects of genome segment modification on viral phenotype which, when transferred to the rotavirus and orbivirus genera, should permit the construction of rationally designed vaccine strains, and may also permit the development of reovirus, a virus that multiplies extensively in humans but does not cause disease, as a gene vector with possible applications in gene therapy.

The conditions under which reovirus RNA is infectious are as follows. The 10 ss ST3 genome segment species, transcribed in vitro by reovirus cores, are translated in a rabbit reticulocyte lysate (RRL) for 60 min. This primed RRL (PRRL) is then lipofected into mouse L fibroblasts together with an equal amount of untranslated ssRNA, and 4-8 h later the cells are then infected with a helper reovirus, either reovirus ST1 or reovirus ST2. 24-48 h later the cells are then harvested, and the virus yield is plaqued on mouse L fibroblasts. If the helper virus is ST2, the plaques are counted after 5 days, well before ST2 plaques develop; if the helper virus is ST1, a 1/1000 dilution of antiserum against ST1 virus is incorporated into the agar plates used for the titration, which completely prevents the formation of ST1 virus. In either case, only infectious ST3 virus is detected. Under standard conditions between 1 and 2 times 10^3 plaques of homologous ST3 reovirus are obtained from 10^6 mouse L fibroblasts. If dsRNA is used and ssRNA in the RRL, the virus yield is about 1-2 times 10^4 plaques; and if ssRNA and dsRNA are used together, with ssRNA in the RRL, the yield is 1-2 times 10^5. If the PRRL is omitted, the yield of virus is 2-3 logs lower; and the same is true if melted dsRNA is used in the RRL rather than ssRNA. It turns out that the essential component formed in the PRRL is not the 10 species of reovirus protein; rather it is RNA-protein complexes, because the potentiating power of PRRLs is destroyed by RNase. Thus the reason why translated melted dsRNA is only very poorly effective (although it is translated very well) is most likely that during the time it takes to lipofect the PRRL into cells, the minus strands rehybridize with plus strands that are components of the active ssRNA-protein complexes, and this rehybridization inactivates them. It also appears that all 10 species of such complexes are required; certainly omission of any of the three size classes of reovirus ssRNA species (l, m, or s) renders the PRRL inactive.

Further features of this infectious reovirus RNA system are as follows. (i) Either ssRNA or dsRNA is infectious (dsRNA 10 times more so) provided that the RRL is primed with ssRNA in both cases. In mixtures of both ssRNA and dsRNA it is the ssRNA that provides most (about 80%) of the material for the generation of progeny genomes, that is, the dsRNA activates the ssRNA. (ii) Genome segment reassortment between the progeny of infectious RNA and ST2 helper virus does not occur to a significant extent (less than 0.1% of progeny are reassortants). The reason for this is not clear but is probably related to the very small yields of infectious ST3 virus formed (on the order of one infectious virus particle per cell, about 1000 times less than the yield of helper virus). Reassortment does, however, proceed in cells lipofected with infectious RNA; when mixtures of ST3 and ST1 RNA are lipofected into cells, about 15% of the yield are reassortants, all of them monoreassortants.

In order for this infectious RNA system to become a system for investigating assortment, encapsidation, etc. signals; functional protein domains; and the construction of virus strains with desirable characteristics, it must provide the means for incorporating novel, that is mutated or modified or heterologous, segments of RNA into the viral genome. However, it has proved impossible to incorporate ST1 or ST2 genome segments into the ST3 genome, using less than their full complement of 10 genome segments. The same is true for all other ST combinations. The only novel products that have been observed (at low frequency) have been virus particles that contain 11 genome segments, and these particles lost the heterologous genome segment within two rounds of replaquing. Remarkably, it has also been found that the infectious reovirus RNA system is incapable of exchanging the genome segments of ts mutants that bear the ts mutation for the corresponding wt genome segment. Thus whereas l + m + s ssRNA yields virus yields wt, that is ts virus; and l + m + s ssRNA of ts447, the ts lesion of which is in the S2 genome segment (6) (49), yields virus capable of multiplying at 32.5, but not at 38.5; the combination of l + m + s + s does not yield ts virus (whereas l + m + s or l + m + s do so). Yet as pointed out above, dual infection of cells with two ts mutants (with mutations in different genome segments) occurs readily (6) .

In order to understand why the replacement of ts by ts genome segments occurs readily in cells infected with virions but not in cells lipofected with genome segments, it is necessary to examine in some detail (i) what, in addition to genome segments, is introduced into cells when cells are infected with virus particles and (ii) the nature of the genome segments present in reassortant virus particles.

With respect to the first issue, infecting virus particles are taken up into cells within endoplasmic vesicles that fuse with lysosomes, in which they lose part, but not all, of their outer shell; they lose proteins 1 and 3 as well as the approximately 100 C-terminal amino acids of protein µ1C(36, 37, 38) . What is liberated into the cytoplasm are subviral particles that are cores covered by a layer of protein , the truncated protein µ1C. As pointed out above, dual infection with infectious virus particles leads to the formation of a full spectrum of multireassortants; lipofection of cells with cores alone, that is with no protein in cells, leads to the formation of only monoreassortants; and lipofection of cells with naked genome segments permits only assortment, not reassortment, of genome segments. What then is the nature of the genome segments of reassortants?


The Nature of the Genome Segments in Reovirus Reassortants

Although reovirus reassortants are rare in nature (all prototype reovirus strains possess homologous genome segment sets; see also (39) for rotaviruses), perhaps because they are less stable than virions encoded by the three homologous genome segment sets or because they multiply more slowly, there are several lines of evidence that some monoreassortants can compete very successfully with viruses containing homologous genome segment sets. The first such line derives from evolutionary considerations.

Whereas for the ST2:ST1 and ST2:ST3 pairs the third base codon divergence percentages are essentially the same for all genome segments (78 ± 4%), these percentages differ enormously among the genome segments of the more recently separated ST1:ST3 pair (for example, for the L3 genome segment there is only 6% divergence in third base codon positions; for L1, 13%; for S4, 22%; for L2, 29%; for S3, 48%; for M2, 53%; for S2, 56%; and for S1, 83%(8, 40, 41, 42) ). The most likely explanation of these widely differing extents of divergence toward randomness is that divergence started at different points in time for each genome segment because the precursor in each case was a reassortant in which an ST1 or ST3 genome segment had become stably associated with the heterologous genome segment set, that is, reassortants were generated at various points in time for each genome segment, when the protein that it encoded exercised its function within the context of the heterologous genome segment set at least as efficiently as the protein encoded by the homologous genome segment. Presumably the L3 genome segments in today's Lang (ST1) and Dearing (ST3) strains of reovirus are derived from a reassortant that emerged rather recently, and the other genome segments are derived from reassortants that arose progressively longer ago(8) .

Another line of evidence derives from the observation that the various MRs that possess 9 ST1 genome segments and 1 ST3 genome segment differ enormously in their ``competitive survival ability.'' In cells dually infected with the S1 MR and wt virus, all progeny virus is the MR; in cells dually infected with the S1 MR and L1 MR, all progeny is the S1 MR and there are no reassortants; and in cells dually infected with the S1 MR and the M1 MR, all progeny is either the S1 MR or reassortants. By contrast, in cells dually infected with the L1 MR and the M1 MR, the progeny consists of equal amounts of each MR and 18% reassortants (equal amounts of wt and double reassortants). Thus some genome segment complements are very much more successful than others, at least with respect to their ability to maintain their identity during multiplication in cultured cells.

In our efforts to examine more closely the reasons for such different competitive survival advantages, the s size class genome segments of the four monoreassortants that contain 9 ST3 genome segments and an S1, S2, S3, or S4 ST2 genome segment were sequenced. The heterologous S1, S2, and S3 genome segments possess no mutations, but in each MR the ST3 S4 genome segment possesses two mutations, G A and G A. Interestingly, these are the same two mutations as are also present in the S4 genome segment of ST3 DI (defective interfering) particles, which are monoreassortants in which the normal L1 genome segment is replaced by a truncated version of it from which its central 2300 bp (out of 3900 bp) have been deleted.

Further, the mutation of G A abolishes an Mln1 restriction site and is therefore readily screened by digesting appropriate polymerase chain reaction-amplified cloned portions of the S4 genome segment. Using this rapid screening technique, it has been found that the ST3 S4 genome segments of all MRs with 9 ST3 genome segments and 1 ST2 genome segment possess the identical G A mutation, as do those of all ST3-ST1 reassortants that possess six or more ST3 genome segments; and that this mutation is barely detectable in stocks of wt ST3 reovirus.

However, this is not the whole story of MR generation because in the MR set containing 9 ST1 genome segments and 1 ST3 genome segment the pattern is different. Here there are no G A and G A mutations in the S4 genome segment of the S1, S2, and S3 MRs, but there are mutations in each reassorted heterologous genome segment, and in each case the effect of the mutations (from one to four mutations) is to render the inserted heterologous genome segment more similar to the ST1 genome segment it replaces. As for the inserted heterologous ST3 S4 genome segment, it also contains a mutation, which is different from either of those in the ST3 S4 genome segment of the first MR set, but which again renders it more similar to the ST1 S4 genome segment that it replaces.

Finally, there is the problem of the failure of the ts S2 genome segment to be reassorted into the ts447 genome segment set in the infectious reovirus RNA system (see above). When the s size class genome segments of ts447 were sequenced, it was found that the S3 and S4 genome segments had no mutations; the S2 genome segment, which encodes the ts 2 protein, possessed the three mutations identified previously(43) ; and the S1 genome segment possessed two mutations, namely C U (A to V) and G A (V to I). Since the l and m size class genome segments possess no mutations precluding reassortment, it is clear that it is these two mutations that prevent acceptance of the ts S2 genome segment into the ts genome segment set.


What Is the Significance of Mutations in Reovirus Reassortants?

Reovirus genome segment reassortment is clearly a complex phenomenon. On the one hand, vastly different genome segments are readily accepted, like the ST2 S1 genome segment into the ST3 genome; it and the ST3 S1 genome segment have diverged 59% toward randomness in their second base codon positions(8) . There is, however, a requirement for the exchange to be effected: and that is two mutations in the ST3 S4 genome segment. These same two mutations are required for the acceptance of all ST2 genome segments introductions into the ST3 genome, including that of the ST2 M2 genome segment, the second base codon position of which has diverged to randomness from that of the ST3 M2 genome segment by only 2%(8) . This suggests that what is needed here is not mutations at the RNA level in order to permit RNA species to recognize each other, but rather generation of a novel protein 3 (which is a component of both ssRCCs and dsRCCs (see above)). However, this does not apply to the 9 ST1-1 ST3 MR system, where heterologous genome segments are accepted with one to four mutations that render them more similar to the genome segment that they replace, but without the G and G mutations in the ST1 S4 genome segment. Nor does it apply to the ts447 system, where two mutations in the S1 genome segment are sufficient to prevent the replacement of the ts S2 genome segment by the ts S2 genome segment, which differs from it in only three loci.

These findings raise several important issues. For example, why are the same ST3 S4 genome segment mutations required in all ST3-ST2 reassortants, which suggests that an altered protein 3 is required, whereas in the ST1-ST3 system mutations in the heterologous genome segments are required? And, why is assortment of vastly different genome segments possible in cells infected with virus particles, but in cells lipofected with genome segments two mutations in a seemingly irrelevant genome segment (S1) are sufficient to prevent the replacement of the ts S2 genome segment by the ts S2 genome segment?

The answers to these questions will hopefully be provided through a comparison of reassortment events in cells infected with the infectious virus particles, with the cores, and with the genome segments of two different parents.


FOOTNOTES

*
Work from the author's laboratory was supported by United States Public Health Service Research Grant RO1 AI 08909 and Research Training Grants T32 AI 07148 and T32 CA 09111 and by Research Grant 3233 from the Council for Tobacco Research. This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995.

(^1)
The abbreviations used are: ts, temperature sensitive; ts, temperature resistant (wild type); wt, wild type; ss, single stranded; ds, double stranded; MR, monoreassortant; bp, base pair(s); RI, replicative intermediate; ssRCC, ssRNA-containing complex; dsRCC, dsRNA-containing complex; RRL, rabbit reticulocyte lysate; PRRL, primed RRL.


ACKNOWLEDGEMENTS

We thank Charles Lin and Igor Nepluev for sharing unpublished results and helpful discussions.


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