Group A rotaviruses (RV), members of the
Reoviridae family, are a major cause of infantile viral gastroenteritis and are responsible for up to 700,000 deaths each year (
6,
24). The RV genome consists of 11 segments of double-stranded RNA (dsRNA) which can be separated by polyacrylamide gel electrophoresis (PAGE), resulting in typical dsRNA profiles exhibiting four well-defined size classes of segments. However, some RV show unusual dsRNA profiles in which standard-sized segments are replaced by larger, rearranged forms (for a review, see reference
5). Gene rearrangements were first reported for RV isolated from immunodeficient children with chronic infection (
11,
26) and can be obtained experimentally by serial passages at a high multiplicity of infection (MOI) in cell culture (
10,
14,
21). We recently showed that rearrangements also occur during acute RV infection (
36). Gene rearrangement usually consists of a partial head-to-tail duplication of a segment sequence. In most cases, sequence duplication occurs after the stop codon, leaving the open reading frame (ORF) untouched and the encoded protein unchanged (
5,
6). Less frequently, the duplication occurs within the ORF, which thus encodes a modified protein (
8,
38).
Manipulation at the cDNA level of most positive- and negative-strand RNA viral genomes, followed by rescue of infectious virus, is now well established and has provided a better understanding of RNA virus replication and pathogenesis. In the case of
Reoviridae, the development of reverse genetics systems has been hampered by the nature of the genome, which carries 10 to 12 dsRNA segments that are densely packed within the viral particle and are transcribed and replicated within a subviral structure. Recent major advances were obtained in the development of reverse genetics systems for some
Reoviridae. For mammalian orthoreo- and orbiviruses, reverse genetics systems based on the transfection of plasmid cDNAs (
13) or cDNA-derived mRNAs (
2) corresponding to a complete set of 10 RNA segments have been established, allowing the rescue of infectious viral progeny. However, for RV, there is no report indicating that transfection of a complete set of viral mRNAs can result in the rescue of infectious viruses, and it is still unknown whether the RV genome can be infectious. In 2006, Komoto et al. (
16) described the first reverse genetics system for RV, based on a model originally developed for influenza viruses by Palese and colleagues (
17). In this system, an exogenous RV mRNA synthesized in the cell cytoplasm by the T7 RNA polymerase (T7pol), expressed by a recombinant vaccinia virus, is packaged in place of its homologous counterpart into a helper RV. This reverse genetics system has allowed the recovery of engineered monoreassortant infectious RV (designated recombinant RV) with an incorporated exogenous segment 4 encoding the spike protein VP4. An
in vitro-modified cDNA-derived segment 4 was also successfully introduced into a recombinant RV to obtain a virus carrying a VP4 antigenic chimera (
15). However, this system is restricted to segments encoding antigenically distinct viral surface proteins (like VP4) because the selection of recombinant viruses requires the use of specific and potent neutralizing antibodies to eliminate wild-type (WT) helper viruses.
Results obtained from a preliminary study suggested that rearranged RNA segments might overcome this limitation. Indeed, we first characterized human RV clones containing rearranged segment 7, 11, or both (
8) and then compared the fitness levels of rearranged versus WT viruses. We found that viruses with rearranged segment 7 or 11 replicated less well than or equally to WT viruses, as judged by viral growth curve experiments. Surprisingly, in competition growth experiments, rearranged segment 7 or 11 was always selected into the viral progenies, even when mixed infections were performed with a ratio of 1 rearranged to 1,000 WT viruses (
4). The absence of a growth advantage conferred on the virus by rearranged segments, combined with their preferential segregation into the viral progenies, suggested that rearranged RNA segments might be packaged preferentially. These observations are in agreement with results of earlier studies showing that rearranged segment 5 or 11 segregated preferentially into viral progenies issued from mixed infections with WT virus (
10,
19).
We developed a reverse genetics system for RV on the basis of the preferential packaging of rearranged RNAs. We report here the rescue of recombinant viruses carrying cDNA-derived rearranged segment 7 (either unmodified or containing silent mutations introduced by site-directed mutagenesis to generate restriction enzyme sites as markers), with no selection pressure other than serial passage in cell culture. We also report the rescue of a recombinant virus expressing a double-sized recombinant NSP3 protein encoded by an in vitro-modified cDNA-derived rearranged segment 7, showing for the first time that an in vitro-engineered gene encoding a modified nonstructural protein can be introduced into an infectious RV.
DISCUSSION
Mammalian orthoreoviruses are the first members of the
Reoviridae family for which RNA was reported to be infectious (
35). In the infectious reovirus RNA system, viral ssRNA, viral dsRNA, and
in vitro-translated viral ssRNA products are transfected (by use of Lipofectamine) together into cells, which are then infected by a helper reovirus of a distinct serotype. Although complex, the system allowed the rescue of temperature-sensitive reovirus mutants, opening the way to
Reoviridae reverse genetics (
31). Indeed, recombinant reoviruses expressing the chloramphenicol acetyltransferase (CAT) protein were obtained by use of this system and a cell line constitutively expressing the product of the engineered gene (
30). In this system, the helper reovirus does not act as an acceptor for exogenous segments to generate engineered reassortants, and its role is not completely understood. More recently, new helper virus-independent reverse genetics systems have been established for mammalian orthoreoviruses and bluetongue virus (BTV), two members of the
Reoviridae family. The strategies are based on the transfection of a complete set of plasmid cDNAs or plasmid cDNA-derived mRNAs that allow for the rescue of recombinant infectious viruses (
2,
13). It is essential for such systems that the complete set of segmented viral RNAs can be infectious when transfected into cells permissive for viral replication. There is no report that infectious RV have been recovered in any cellular system from RNAs produced by viral cores, plasmid cDNAs, or plasmid cDNA-derived mRNAs, and the use of a helper RV to generate engineered reassortants (i.e., recombinant for the exogenous segments) is still mandatory, as described here and by Komoto et al. (
15,
16). Our results confirm the feasibility of integrating an exogenous segment into an infectious RV, as reported by Komoto et al. for segment 4. Additionally, our findings indicate that although initial recombination events probably occur at a low frequency, the capacity of rearranged segments to be packaged preferentially over WT segments is efficient enough to support the growth of recombinant over WT viruses.
Results obtained during optimization experiments showed that vaccine infection and timing of plasmid transfection can drastically affect the permissiveness of COS-7 cells for RV infection, possibly related to some changes in cell membrane properties. Consequently, only 10% of COS-7 cells could actually be both transfected by a pRiboz plasmid and coinfected by vaccinia virus and RV, and the proportion of recombinant RV carrying an exogenous RNA segment should be infinitesimally low among the viral progenies produced in COS-7 cells. We previously reported that in a mixture of viruses with WT and rearranged segments 11, the rearranged segments could be detected by RT-PCR if they were present at a threshold ratio as low as 1 rearranged segment to 10
4 WT segments and by PAGE for a ratio as high as 1 to 1 (
36). Similarly, the RT-PCR assay we used here was able to detect a rearranged segment 7 at a threshold ratio of 1 rearranged to 10
5 WT segments 7. Considering these detection thresholds, we can estimate that the ratio of recombinant to WT viruses was approximately 1:10
5 at passage 3 (first positive RT-PCR detection) and increased 10-fold every 2 or 3 passages, to reach a ratio of 1:1 at passage 15 to 18 (first positive PAGE detection). We can thus estimate that in the viral progeny produced by COS-7 cells, the ratio of recombinant to WT viruses was ≤1:10
6. In addition to this low ratio, it may be important to consider the impact of the MOI used for further serial passage in MA-104 cell culture on favoring the recovery of recombinant viruses. Indeed, it has been reported that in mixed infections of WT bovine RV and bovine RV with a rearranged segment 5, viruses with a rearranged genome overgrew during passage at a high MOI (undiluted inoculum), whereas WT virus overgrew during passage at a low MOI (1:100-diluted inoculum) (
10). In this study, undiluted inoculum was used for serial passage, and recombinant viruses were recovered after a period of 5 to 6 weeks (15 to 18 cell passages, with 3 passages per week). This time lag could be shortened with the help of RT-PCR detection, and only 1 week (3 passages) was required to ascertain that recombinant viruses had actually been generated, improving the feasibility of the system. We are currently working on further improving the efficiency by RT-PCR testing for recombinant viruses in plaques or pools of plaques obtained from earlier cell culture passages.
To rescue recombinant RV expressing genetically engineered VP4 proteins (
15,
16), Komoto et al. relied on the use of potent neutralizing antibodies, thus limiting the system to genes encoding surface capsid proteins. Here we show that distinct forms of exogenous rearranged segment 7 can be rescued by propagating the viral progeny in cell culture without any additional selective pressure. We set up our reverse genetics system by using rearranged segments 7 with a bovine RV as a helper virus, but whether this system can be extended to other rearranged segments or to different strains of helper viruses remains to be determined. In particular, this system might not apply to helper viruses that grow poorly in cell culture, like most human RV, or to segments for which rearrangements have not yet been identified in viable RV. Infectious RV carrying one or more rearranged segments have been described for 7 of the 11 genomic RNA segments (segments 5 to 11) (
5), and it has been shown that rearranged segment 5 or 11 segregated preferentially to the homologous WT counterpart in viral progenies issued from mixed infections (
10,
19). It is thus reasonable to hypothesize that our system should allow the production of viable recombinant viruses for at least these seven segments and should extend the possibilities of RV reverse genetics to gene segments encoding viral proteins (such as NSP3) for which potent selection tools (such as neutralizing antibodies) are not and will not be available. Because of steric constraints due to the huge concentration of viral dsRNA inside the viral particle, the length or number of rearranged segments could be a limiting factor for packaging. We previously described an RV carrying two rearranged segments, segments 7 and 11, corresponding to 1,531 additional bp packaged into the virus (
8). RV can package as many as 1,800 additional bp, i.e., approximately 10% of the standard genome, and RV containing three rearranged segments with partially duplicated sequences have been reported (
5,
11,
20). Similarly, it has been shown that the capacity of reovirus to package additional genetic material is probably limited to 2 kb, which is also about 10% of the whole genome (
33). The lengths of RNA segments may also interfere with RNA replication. Skehel and Joklik reported that the relative number of RNA transcripts obtained from reovirus cores was inversely proportional to their molecular weight (
37). In a study using a cell-free RV replication system, Patton et al. showed that the size of the RNA template is an important factor affecting the synthesis of dsRNA by open cores, with an inverse relationship between RNA length and replication efficiency (
25). Interestingly, this was not the case for a rearranged RNA, which was 1.5-fold longer than the WT RNA but was replicated with a similar efficiency. Thus, rearrangement of RNA does not seem to affect RNA replication. This is important to consider, since one limitation of our reverse genetics system is that all generated recombinant viruses will contain a rearranged segment. However, to study protein function by mutagenesis of the ORF carried by a rearranged segment, it will be necessary to compare the mutated to the unmutated ORF in the context of the same long 3′-UTR generated by the rearrangement for assignment of changes in virus phenotype to ORF modification.
We show here for the first time that an
in vitro-engineered gene 7 encoding a modified NSP3 protein can be introduced into an infectious RV. Three functional and structural domains have been established for NSP3 (
3,
27,
28): the N-terminal domain (aa 1 to 150), which specifically binds to the 3′ end of viral mRNAs; the central domain (aa 150 to 241), which contains a coiled-coil domain required for dimerization; and the C-terminal domain (aa 206 to 313), responsible for interaction with the eIF4G translation initiation factors. It has been established that during RV infection, NSP3 evicts PABP from eIF4GI, leading to a concomitant shutoff of cellular mRNA translation (
28,
39,
40). However, enhancement of RV mRNA translation by NSP3 was recently disputed, on the basis of RNA interference experiments showing that NSP3 knockdown has limited effects on viral protein synthesis and viral production in cell culture (
22). Our reverse genetics system makes it possible to introduce specific mutations into the NSP3 gene that will be useful for specifying NSP3 functions during the viral replication cycle. Note that recombinant viruses expressing the modified r-NSP3 protein have a small-plaque phenotype. Since it has been suggested that plaque size directly correlates with the degree of suppression of host cell mRNA translation (
10,
11), this small-plaque phenotype may indicate a change in the NSP3 function related to the shutoff of cellular protein synthesis. One hypothesis could be that duplication of binding domains of the r-NSP3 protein may interfere with some host factor.
The reasons that RV rearranged segments are preferentially packaged into viruses remain to be determined. One could hypothesize that duplication of some packaging signals in rearranged segments may double their probability of being encapsidated. Packaging signals have not yet been identified for RV. In this respect, comparison between homologous rearranged segments from different RV strains may tentatively identify conserved duplicated sequences and/or conserved duplicated stem or stem-loop secondary structures possibly involved in packaging. For reovirus, the existence of packaging signals was first suggested by the ability to package subgenomic S1 segments into defective interfering particles (
23). More recently, Roner et al. identified, with the help of reverse genetics, the packaging signals contained in the 5′- and 3′-terminal sequences of the L1, M1, and S2 reovirus genes. The minimal 5′ and 3′ sequences required for packaging range from 96 to 129 nt and from 98 to 173 nt, respectively, extending across the coding sequences. Additionally, the nucleotides used to identify the ssRNAs are localized to the 5′, not the 3′, termini (
29,
32-
34). The
cis-acting sequences required for packaging of the BTV VP6 gene have also been shown to overlap coding sequences (
18). For RV, it has also been suggested that a
cis-acting structure essential for packaging might exist outside the 5′ and 3′ ends (
9). It is interesting that in the RV rearranged segment 7 we used, the 5′ part of the coding sequence is fully or almost fully duplicated, while the 3′-untranslated terminus is unique. This might suggest that, as for reovirus, RV packaging sequences are not in the 3′ terminus and reach further inside the 5′ part of the gene. Similarly to the reovirus model, our reverse genetics system should allow testing of mutants deleted for distinct parts of the duplicated sequence in order to specify (at least for gene 7) the minimal sequence essential for preferential packaging. As opposed to the reovirus or BTV system, which requires a cell line constitutively expressing the product of the gene that is tested for packaging by deletion analysis (
18), a system using RV rearranged segments deleted for noncoding duplicated sequences does not require complementation. Characterization of the minimal sequence essential for preferential packaging could allow us to shorten the sequence duplication in engineered segment cDNAs and to optimize vector size for reverse genetics experiments. This will be most useful for the manipulation of the large genomic RNA segments 1 to 4 or for incorporating a set of several exogenous segments together. Thus, by engineering partial gene duplication at the cDNA level, reverse genetics could be possible for virtually every RV RNA segment.
In conclusion, the reverse genetics system for RV described here opens new possibilities for investigating RV protein functions, particularly those linked to virulence and pathogenesis. A better understanding of RV attenuation mechanisms would be helpful for the development of recombinant live RV vaccines containing targeted changes for attenuation.