Recovery of intraspecies C recombinants from co-transfections
Both intra- and interserotypic PV recombinants have being reported, and a few intraspecies crosses between PV and species C viruses have been identified in
Figure 4.4 Virus recovered from BsrT7 cells co-transfected with PV1 and PV3 RNA partners. (A) Crystal violet stained HeLa cells infected with undiluted transfection supernatant and overlaid with plaque assay overlay medium. Control wells containing one RNA partner were clear; plaques were present at ratios 1:1 and 1:4 of combined RNA partners. (B) Schematic of PV3/PV1 recombinants generated by co-transfection, with RdRp template switching occurring between luciferase and mutated CRE (within the shaded region).
clinical isolates (Kew et al., 2002; Rakoto-Andrianarivelo et al., 2007). However, widespread intraspecies recombination has been described for other members of enteroviruses, justifying the need to attempt similar experiments using the method developed in this study (Oberste et al., 2004a; Oprisan et al., 2002; Santti et al., 1999; Simmonds & Welch, 2006).
To examine whether this model could yield intraspecies recombinants, the available PV subgenomic replicons were co-transfected with artificially constructed
recombinants containing coxsackievirus A (CVA)21 non-structural regions. A series of full-length, intraspecies recombinant clones containing CVA21 with PV1 or PV3 segments were constructed by Dr. Claire Blanchard using basic molecular cloning techniques during her PhD (figure 4.5). The plasmids CVA21BKSabin1P1 and CVA21BKPV3P1 contained a PV1 and PV3 capsid respectively, in a pRiboCVA21 backbone. Plasmid PV3BKCVA21P1 was constructed by introducing a CVA21 encoded capsid into a PV3 backbone vector derived from pT7FLC/REP3 (Barclay et al., 1998). Exchanges of P1 sequence were performed whilst retaining
phenylalanine/glycine (in the case of CVA21) and tyrosine/glycine (in the case of PV3 and PV Sabin-1) amino acid sequences at the VP1/2A junctions to maintain correct processing (Claire Blanchard’s thesis, University of Reading, 2004). Interestingly, the transfected chimeric T7 RNA transcripts did not cause CPE in HeLa or RD-ICAM cells meaning that they did not produce viable virus. VP1 protein expression was detected in cells transfected with RNA derived from recombinant cDNAs indicating correct translation and processing by heterologous proteases of RNA. Although all constructs translated and processed-proteins correctly (as shown by in vitro and invivo transcription/translation assays), they were not capable of producing infectious virus, explained by possible defects in virus maturation, assembly, or possible lack of compatible packaging signals. The constructed recombinants appeared to be replication-competent, even though levels of newly synthesised RNA were not as high as those observed in positive control samples. As a CVA21 replicon and full-length CVA21 CRE mutant were not available during the current project, the aforementioned clones provided replication-competent CVA21 RNA for co-transfection experiments with PV RNA using this model. Restriction sites used to linearise plasmids are outlined in table 2.2. The three constructs were
Figure 4.5 Intraspecies C recombinants constructed by Dr. Claire Blanchard. The genomic origin of recombinants constructed previously – the capsid sequence has been removed from the full-length infectious cDNA and replaced with another species C virus capsid sequence.
co-transfected with either pRLucWT (PV1) or pT7Rep3-L (PV3) subgenomic replicons.
Using similar assay conditions to those employed previously, a total of 2 µg of 1:1 RNA partners was co-transfected into L929 or BsrT7 cells. Likewise, individual partner RNA was also transfected into cells to confirm their inability to create viable virus alone. Supernatant was harvested 48 hrs post-transfection and inoculated undiluted onto HeLa cells or RD-ICAM cells in the case of reactions containing CVA21 coding capsid regions. RD-ICAM cells are modified RD cells expressing ICAM-1 receptor required by CVA21 for cell attachment and infection.
Figure 4.6 illustrates results of plaque assays in HeLa cells only. Each RNA partner transfected into cells alone did not produce virus as indicated by clear HeLa
monolayers – neither the replicons nor constructs were able to generate virus on their own.
However, virus was recovered from the following co-transfections: pRLucWT + CVA21BKPV3P1, pRLucWT + CVA21BKSabin1P1, pT7Rep3-L +
CVA21BKPV3P1, and pT7Rep3-L + CVA21BKSabin1P1 (figure 4.6). No CPE was evident with RD-ICAM cells when supernatant was harvested from cells co-
transfected with either replicon together with PV3BKCVA21P1 (data not shown). It was not surprising that virus was not recovered from these co-transfections, as it was shown previously that chimeras generated with CVA (species C) capsids and the replication proteins of PVs were either dead or weakened (Jiang et al., 2007). This phenomenon is discussed in more detail at the end of this chapter.
Virus containing supernatants harvested from pRLucWT + CVA21BKSabin1P1 and pT7Rep3-L + CVA21BKPV3P1 co-transfections were retained for future
investigation. Recovered viruses were confirmed as recombinants by sequencing and crossover junctions between RNA partners were characterised and are discussed in detail in chapter five.
Figure 4.6 Virus recovered from intraspecies RNA co-transfections. These combined PV subgenomic replicons and Dr. Blanchard’s constructed intraspecies recombinants (CVA21BKSabin1P1, CVA21BKPV3P1, and PV3BKCVA21P1). Transfection supernatant was plated undiluted onto a HeLa monolayer and overlaid with plaque overlay medium. Cells were stained 48 hrs post-infection. All control wells with one RNA partner only were negative. Plaques are evident in co-