CHAPTER 2: Materials and Methods 42
3.10 Discussion 93
Recombination is a well-‐defined phenomenon in enteroviruses, as well as other important positive-‐strand RNA virus pathogens. However, the underlying mechanism(s) by which recombinants arise is relatively poorly understood. Studies on replicative recombination have suggested that the copy-‐choice template-‐switching model is additionally influenced by
sequence homology between parental genomes, or by the RNA secondary structure of donor or recipient templates (section 1.3).
The analysis of historical isolates has generally implied that the selected recombinant genome contains a junction that reflects the point at which the polymerase switched from donor to recipient template. The development of a novel in vitro assay that generates recombinant enteroviruses in the absence of parental virus background allows the isolation of recombinants soon after they have arisen. This minimises additional selection and competition that may occur following serial passage. The experimental evidence gained has provided some interesting results to debate.
A previous study using this approach (Lowry et al., 2014) has shown that the primary progeny from intertypic recombination are largely ‘imprecise’ in nature, with clustering of junctions primarily found within two main gene boundaries: VP1/2A and 2A/2B. The consequences of this clustering are that the majority of imprecise recombinants retained the ability to encode non-‐chimeric versions of the 2A and 2B proteins. This clustering maybe a consequence of the overriding requirement for functional 2A and 2B proteins in the initial recombinant and this may not occur in a single step. Indeed, functionality at this early stage seems paramount, as an in-‐depth analysis of over 100 intertypic recombinant isolates could find no obvious or clear link between RNA sequence and only a limited link between RNA secondary structure and the site of
recombination (Lowry et al., 2014).
The characterisation of an imprecise intertypic recombinant virus from a co-‐transfection of HeLa cells indicated that the promiscuous nature of recombination was not as a result of a rodent cell specific factor. Only a minimal amount of progeny were sequenced and they could all have arisen from the same recombination event, but the principle of imprecise recombination was shown. The recombinant sequence itself, shown in figure 3.6, suggested a switch from the PV1 replicon template to the acceptor template at an ‘A’ triplet motif. Intertypic poliovirus recombination junctions have been linked to similar sequences in a previous study (King, 1988b).
It has been suggested that the reason why fewer intertypic and interspecies recombinants are observed in nature is due to the sequence divergence between RNA partners (Kirkegaard and Baltimore, 1986b). Using standardised conditions, the CRE-‐REP assay showed that intratypic recombination frequency is significantly higher than intertypic recombination (Chi square test). Indeed, approximately 10-‐fold more frequent, which is a figure slightly less than the 100-‐fold difference previously postulated (Kirkegaard and Baltimore, 1986b, King, 1988b, Tolskaya et al., 1983). Although, the amount of recombinant progeny isolated may be inherently linked to its type. Perhaps, intratypic recombinants replicate better than intertypic recombinants following the initial recombination event due to correct protein-‐to-‐protein interactions. This would affect the overall yield of virus and not necessarily represent the actual number of recombination events. The qualitative data suggested that the type of recombinant, precise or imprecise, could be directly linked to the sequence identity of the parental genomes. In section 3.3, isolation and characterisation of intratypic recombinant virus indicated that only a minor amount were imprecise with additional sequence at the recombination junction (3/20). This contrasts to the generally imprecise intertypic recombinants isolated to date. These findings suggest that sequence identity may indeed influence location of recombination. Although, section 3.6 described the secondary stage of recombination in enteroviruses, a stage we term ‘resolution’.
All three imprecise intertypic recombinants lost all the additional sequence following serial passage in HeLa cells and returned to wild type length. Sequence analysis of the ‘resolved’ isolates indicated new recombination junctions, where the precise location of recombination could not be identified due to sequence identity of the parental genomes. This finding was further supported by the construction of a molecular clone that represented one of the passaged imprecise recombinants. Similarly, this construct went through the resolution process and produced a genome indistinguishable from wild type in length. Sequence analysis also
highlighted sequence identity at the new recombination junctions and provided a junction that was exactly the same as the biological clone that carried the same imprecise sequence. This strongly suggests that the characterisation of intertypic field isolates that has proposed sequence identity at the recombination junction (King, 1988b) have missed the early events of recombination and only characterised the end product. The sequence identity found within the resolved recombination junctions may indicate that sequence does play a role, but perhaps not during the initial stage which seems a more promiscuous process that may be subjected to other primary limitations e.g. packaging restraints and protein-‐protein functionality. It also remains to be determined if the resolution stage of recombination is a cis or trans event that involves the viral RdRp. In principle, it could be either or even both. The fewer imprecise intratypic recombinants isolated may actually be a reflection of their ability to resolve at a faster rate than an imprecise intertypic recombinant. Potentially, the characterised intratypic recombinants in section 3.3 had already gone through the secondary resolution stage where any additional sequence is lost. Indeed, a single poliovirus genome is replicated an average of six times in a single cell (Schulte et al., 2015). Perhaps sequence identity is a key factor during subsequent rounds of replication within the same cell following the initial promiscuous recombination event? To answer this an imprecise intratypic recombinant, similar to JC105B could be produced. Serial passaging of such a recombinant, when compared to an intertypic version (JC105B) might highlight any differences in the resolution process.
The ability to extend the CRE-‐REP assay into a species B enterovirus group indicates that the approach may be universal to these genera of viruses. The inability to isolate intertypic
recombinant (E7/E6) was disappointing. They share similar divergent genomes as that of PV3/PV1 at ~ 20%, indicating their suitability to the assay. As stated, this study has shown that intratypic recombination frequency is 10 fold higher than intertypic, a finding broadly in line with previous studies (Kirkegaard and Baltimore, 1986b). The inability to recover E7/E6 recombinant virus is therefore not surprising given the relatively low frequency of E7/7 recombinants isolated from BsrT7 cells. Potentially the permissive cells that were being used were sub-‐optimal for this species of virus. Unfortunately, due to time restrictions an extended study of this group was not carried out and it was determined that all future experiments would concentrate upon the enterovirus C poliovirus.
The CRE-‐REP assay, in principle, could have produced the initial recombinant virus by either a non-‐replicative or replicative pathway. The development of the NON-‐REP assay that was based on previous non-‐replicative approaches (Gmyl et al., 1999, Gmyl et al., 2003) allowed
quantification of viable recombinants within the same 1058 nt region between the 2C CRE and P1 coding region. This allowed the relative contribution of non-‐replicative recombination to the CRE-‐REP assay to be calculated. The very low yield of recombinant progeny in the NON-‐REP assay when compared to the postulated replicative approach strongly suggested that recombination in the CRE-‐REP assay was primarily replicative.
The suggestion that an imprecise replicative recombination process results in partial duplication of the virus genome also has implications for the wider understanding in the evolution of positive strand RNA viruses. There are several well-‐characterised evolutionary duplication events that have shaped the picornavirus genome. For example, the capsid proteins (VP1-‐3) share a common ‘jelly roll’ structure (Hogle et al., 1985) that have arisen from
duplication. Another example is the tandem repeat regions that encode the three contiguous VPg proteins of foot and mouth disease virus (Forss and Schaller, 1982) and even the non-‐ contiguous 2A and 3C proteases (Palmenberg at al., 2010). In addition, other positive strand RNA viruses exhibit genome duplications (Simon-‐Loriere and Holmes, 2013) such as the
multiple VPg proteins of some dicistroviruses, which can encode as many as six from one genome. It was postulated that the redundancy in VPg coding provided an advantage in overall replication rates (Nakashima and Shibuya, 2006). Additionally, the functionally distinct adjacent leader proteinases of the Closteroviridae, that have arisen from duplication events, provide common protease activities but also possess specialised functions in virus RNA replication, virus invasion and cell-‐to-‐cell movement (Peng et al., 2001). The type of imprecise
recombination event described in section 3.3, potentially coupled with partial resolution, could account for this ‘evolution by duplication’. Likewise, duplication and subsequent resolution events could explain the unique C-‐A-‐B motif arrangement in the palm domain of the polymerase reported in some Alphavirus-‐like insect tetraviruses (Gorbalenya et al., 2002). Other than the ancestral evolution of the capsid proteins the majority of these involve duplication of relatively short sequences, and this is presumed to reflect intrinsic constraints on genome length that may prevent their fixation (Simon-‐Loriere and Holmes, 2013).
In summary, recombination in enteroviruses is a biphasic process that provides an insight into the potential mechanisms of genome evolution. The observations in section 3.9 strongly suggested that the process of recombination in the CRE-‐REP assay was primarily replicative. The characteristics of the viral RdRp (a key factor in the copy-‐choice model of recombination) that determine recombination frequency were therefore considered an area of interest.