Genome scale patterns of recombination between coinfecting vaccinia viruses

Abstract

Recombination plays a critical role in virus evolution. It helps avoid genetic decline and creates novel phenotypes. This promotes survival, and genome sequencing suggests that recombination has facilitated the evolution of human pathogens, including orthopoxviruses such as variola virus. Recombination can also be used to map genes, but although recombinant poxviruses are easily produced in culture, classical attempts to map the vaccinia virus (VACV) genome this way met with little success. We have sequenced recombinants formed when VACV strains TianTan and Dryvax are crossed under different conditions. These were a single round of growth in coinfected cells, five rounds of sequential passage, or recombinants obtained using leporipoxvirus-mediated DNA reactivation. Our studies showed that recombinants contain a patchwork of DNA, with the number of exchanges increasing with passage. Further passage also selected for TianTan DNA and correlated with increased plaque size. The recombinants produced through a single round of coinfection contain a disproportionate number of short conversion tracks (<1 kbp) and exhibited 1 exchange per 12 kbp, close to the ∼1 per 8 kbp in the literature. One by-product of this study was that rare mutations were also detected; VACV replication produces ∼1×10(-8) mutation per nucleotide copied per cycle of replication and ∼1 large (21 kbp) deletion per 70 rounds of passage. Viruses produced using DNA reactivation appeared no different from recombinants produced using ordinary methods. An attractive feature of this approach is that when it is combined with selection for a particular phenotype, it provides a way of mapping and dissecting more complex virus traits.

Importance: When two closely related viruses coinfect the same cell, they can swap genetic information through a process called recombination. Recombination produces new viruses bearing different combinations of genes, and it plays an important role in virus evolution. Poxviruses are a family of viruses that includes variola (or smallpox) virus, and although poxviruses are known to recombine, no one has previously mapped the patterns of DNAs exchanged between viruses. We coinfected cells with two different vaccinia poxviruses, isolated the progeny, and sequenced them. We show that poxvirus recombination is a very accurate process that assembles viruses containing DNA copied from both parents. In a single round of infection, DNA is swapped back and forth ∼18 times per genome to make recombinant viruses that are a mosaic of the two parental DNAs. This mixes many different genes in complex combinations and illustrates how recombination can produce viruses with greatly altered disease potential.

FIG 1

Patterns of DNA exchange in recombinant vaccinia viruses. The genome sequences of DTM (A) and DTH (B) recombinant clones were aligned against the parent genomes DPP17 and TP05 using the program LAGAN and edited using the program Base-By-Base. TP05 was used as the reference strain, and any differences between a given virus and TP05 are color coded to indicate insertion, substitution, and deletion mutations derived from strain DPP17. Thus, the blank regions represented fragments derived from TP05.

FIG 2

Rare mutations associated with replication and recombination. (A) Deletion mutation in DTM29, immediately adjacent to a G-to-T SNP found at alignment position 70493; (B) C-to-T substitution detected only in clone DTM27 at position 900.

FIG 3

Numbers of exchanges in DTM and DTH clones. Each of the hybrid viruses was first aligned against strains TP05 and DPP17. Then, the program Base-By-Base was used to determine where each crossover was located relative to the 1,399 SNPs that differentiate the two strains, along with the number of such exchanges. The viruses passaged five times (DTM) exhibited more exchanges per genome than the viruses passed just once (DTH), that is, 30 ± 11 versus 18 ± 11 exchanges/genome, respectively.

FIG 4

Lengths of the DNA segments exchanged in DTH clones. The lengths of all the conversion tracks were measured in all 14 genomes using midpoints defined by the four SNPs flanking the two bounding sites of exchange. The numbers of events of a given exchange length were then determined by assignment to 200-bp bins. A semilogarithm of the bin size (i.e., exchange length) is presented because the values differ so greatly in scale across the different genomes. VACV recombination appears to be associated with a disproportionate number of very short exchanges. Because there is approximately one SNP per 140 bp, resolution greater than ∼200 bp is not achievable.

FIG 5

Biased selection for sequences associated with the TianTan parent. The percentage of DNA derived from each of the parental viruses was determined from the fraction of SNPs derived from each parent. Panel A shows how the composition varied in viruses passaged just once (DTH hybrids) or five times (DTM hybrids) prior to cloning. Passage appeared to select for SNPs linked to the TP05 TianTan parent, as the percentage of Dryvax DNA decreased from 50% ± 27% to 19% ± 11% with continued passage. Panel B illustrates how the plaque size is related to the genetic origins of the hybrid. The viruses forming smaller plaques more closely resemble the DPP17 parent. To measure the plaque size, each of the cloned hybrids was plated on BSC-40 cells (in parallel), cultured for 2 days, stained with crystal violet, and scanned, and the plaque area was determined using ImageJ (24). Twenty randomly selected plaques were measured for each virus.

FIG 6

Illegitimate recombination detected during the cloning and sequencing of hybrid DTM28. During the sequencing of DTM28, 11 reads were detected linking gene DVX_201 to gene DVX_239. Panel A shows an alignment of these reads to sequences within the two genes, which are normally spaced 21 kbp apart. We have also identified sequence identities (circles), short patches of homology (boxed), and a simple repeat (underlined) common to sequences flanking the fusion site. The sequence in these reads transitions cleanly from one gene to the next, with no evidence of any unrelated additional sequences having been added in the process. Panel B showed a way to form this deletion. Illegitimate recombination between identical parents (DTM28) excised 21 kbp and created the virus we subsequently called DTM28Δ. Panel C shows the results of a PCR analysis using primers targeting sites flanking the fusion site. These are located too far apart in the parent viruses (e.g., DTM27) to amplify 21 kbp of intervening sequence. The DTM28Δ virus was probably formed during the expansion of the clone prior to sequencing, as it is not detected in intermediary viruses during the course of passages. Panel D shows a Southern blot of NdeI-digested virus DNA showing that the sequenced virus stock contained two viruses. These are the DTM28 hybrid (indicated by a 5.4-kbp fragment common to both parent strains), and DTM28Δ (indicated by a 6.7-kbp fragment containing the fusion junction). Panel E shows that DTM28Δ is independently viable. Six randomly selected viruses were separately plaque purified from the sequenced stock, and PCR was used to detect sequences found only in the deleted region in DTM28 (primers 208F + 209R) or capable of being amplified only if the intervening sequences are deleted (primers 201F + 239R).

FIG 7

Patterns of DNA exchange in recombinant vaccinia viruses produced using leporipoxvirus-mediated reactivation reactions. The genome sequences of the DTD recombinant clones were aligned against the parent genomes DPP17 and TP03 using the program LAGAN and edited using the program Base-By-Base. Because this experiment used TP03 DNA, and TP05 was always used as the reference strain in all of our analyses (Fig. 1), the telomeric deletion mutations that differentiate TP05 from TP03 show up as additional red blocks in the TP03 alignment.