The replacement of the HRV-A16 5‟UTR by those of HRV-B14, an HRV-C11 strain (HRV-Ca subspecies), an HRV-Cc strain (GenBank accession no. JN087518) and even of HEV-A71 resulted in viable viruses. This suggests that the 5‟UTR is largely interchangeable between different HRV species, and even between HRVs and HEVs. An extended number of 5‟UTR-ORF chimeras should be tested to assess to what extent this statement is true. Nevertheless, these results are congruent with identified sites of natural HRV recombination close to the 5‟UTR-ORF junction [67, 69]. In addition, the 5‟UTR region is the most conserved genomic region among HRVs and cannot be used to segregate HRV types into species, which may explain why interspecies recombination is possible in this region.
Interestingly, the fittest interspecies recombinant harboured the HEV-A 5‟UTR, whose sequence is the most divergent to that of the HRV-A16 5‟UTR among the chimeras used in this study. This finding could be explained by the fact that the biologic activity of secondary RNA structures of 5‟UTR elements plays a crucial role in HRV translation and replication processes, irrespective of the actual nucleotide sequence. This would imply that the enterovirus 5‟UTR per se is particularly efficient in promoting viral translation and replication. See also the discussion section in Schibler et al [162].
Concerning experiments involving chimeric HRVs in which the coding region for the capsid (VP4, V2, VP3 and VP1) and 2A proteins were replaced with corresponding sequences of
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another HRV genome, only the intra-species HRV-A16/HRV-A81 recombinant gave rise to viable and replication competent virus. None of the HRV-A16/HRV-C11, HRV-C11/HRV-A16 and HRV-HRV-C11/HRV-A16/HRV-B14 interspecies chimeric constructs could be amplified in culture.
Furthermore, immunofluorescence performed 7 days post transfection with an antibody detecting the double-stranded replication intermediate was negative, suggesting that the absence of viability of these interspecies chimeric genomes resides upstream of the viral replication process. Of note, as mentioned in the materials and methods section, the non-recombinant HRV-C genome used for the design of two of our chimeric constructs was unable to replicate upon transfection.
Experimental non replicative HRV RNA recombination
The non replicative RNA recombination approach was first described by Gmyl et al for poliovirus genomes [73]. However, in these experiments, the authors used deleted poliovirus genomes designed to recombine in the 3‟ end of the 5‟UTR. In this study, the co-transfection partners were not engineered to promote recombination in a particular genomic region.
Instead, due to 5‟ and 3‟ terminal deletions, recombination could occur at any position in between. As a proof of principle, co-transfection of a 5‟deleted HRV-A16 genome with a 3‟deleted HRV-A16 genome yielded recombinant viruses in a high proportion of experiments.
The two different 5‟deleted constructs used provided similar results.
As expected, non replicative RNA recombination between HRVs belonging to different types but to the same species was less efficient. Indeed, co-transfection of a 5‟UTR deleted HRV-A16 genome and a 3‟ deleted HRV-A39 genome resulted in viable recombination in two out of eight wells. Repeating these experiments allowed us to recover two additional intraspecies recombinants. Mapping of the recombination sites revealed breakpoints in VP2, VP1 and 3D.
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The identification of recombination sites in the capsid genes was rather surprising, as this has never been described in natural HRV intraspecies recombination. This may be linked to a different recombination mechanism occurring in vivo, namely replicative recombination, or to a specific environmental pressure preventing the emergence of HRVs having recombined in the region coding for capsid proteins. Finally, the parental genomes are not defective in vivo and may outcompete recombinants at the capsid level. Nevertheless, these results suggest that chimeric capsids originating from different serotypes within a same species are potentially functional. As for the artificially engineered chimeras, no inter-species recombinant could be recovered by non replicative recombination. However, we observed clusters of a few IF-positive cells after HRV-C11 del3‟ and HRV-A16 del5‟UTR(1-434) suggesting that viable non replicative RNA recombination did occur, but the resulting virus was not fit enough to be successfully passaged and sequenced. Of note, though these data are in agreement with data obtained with chimeric genomes, solid conclusions are difficult to draw since the full-length HRV-C11 genome was unable to replicate and was initially used in an attempt to understand why HRV-Cs could not be propagated in cell cultures.
Altogether the artificial and non replicative recombinants generated in this study suggest that although 5‟UTR interspecies recombinant are viable, the recombination potential at the polyprotein level is much more limited and possible only within a serotype or to a lesser extent within a given HRV species. These global findings are in agreement with observations derived from circulating HRV recombinants [67].
Interspecies recombination in the polyprotein region did not result in fully infective virions regardless of the experimental approach used. There are at least two constraints that may limit interspecies recombination. First, as polyprotein cleavage sites differ among the three HRV species, a likely hypothesis explaining the absence of viable interspecies HRV recombinants
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at the polyprotein region level is that the 2A and 3C proteases of a given HRV species can only fully process polyproteins translated from genomes of the same species. In experiments performed in this study 2A protease cleavage incompatibilities were avoided by exchanging the 2A gene along with the capsid genes originating from the same HRV genome. Concerning 3C protease cleavage sites, we identified one in the HRV-B14 capsid region that differs from the corresponding site in the HRV–A16 polyprotein, between the VP3 and VP1 regions (Table 3). Hence, this site in the HRV-B14 capsid region might not be appropriately cleaved by the HRV-A16 3C protease, possibly explaining why the HRV-A16/HRV-B14 P12A recombinant did not yield viable virus. Second, the fact that cre elements, essential for HRV replication, are located in different ORF regions according to the different HRV species constitutes another obstacle susceptible to limit interspecies HRV recombination in vivo. A substantial proportion of theoretical interspecies HRV recombination combinations could therefore result in the absence of a functional cre element, rendering the recombinant HRV genome unable to replicate. On the other hand, an additional cre may arise from interspecies HRV recombination, which might interfere with the replication process. Our engineered P1-2A HRV recombinants all displayed a single and functional cre structure, which theoretically allows the replication process to occur properly. However, potential replication disturbances related to the displacement of this element in interspecies P1-2A chimeras cannot be formally excluded.
The absence of interspecies HRV recombination in these two systems is somewhat in contradiction with the HRV interspecies recombinants documented in the literature [69]. This may rely on the fact that all these documented recombination events are ancient and might have occurred before the individual speciation of the parental HRV genomes.
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