Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature.
DelatteH.,
MartinD. P.,
NazeF.,
GoldbachR.,
ReynaudB.,
PeterschmittM.,
LettJ. M.2005; South West Indian Ocean islands tomato begomovirus populations represent a new major monopartite begomovirus group. J Gen Virol 86:1533–1542[CrossRef]
DonsonJ.,
Morris-KrsinichB. A.,
MullineauxP. M.,
BoultonM. I.,
DaviesJ. W.1984; A putative primer for second-strand DNA synthesis of maize streak virus is virion-associated. EMBO J 3:3069–3073
DrummondD. A.,
SilbergJ. J.,
MeyerM. M.,
WilkeC. O.,
ArnoldF. H.2005; On the conservative nature of intragenic recombination. Proc Natl Acad Sci U S A 102:5380–5385[CrossRef]
García-AndrésS.,
MonciF.,
Navas-CastilloJ.,
MorionesE.2006; Begomovirus genetic diversity in the native plant reservoir Solanum nigrum : evidence for the presence of a new virus species of recombinant nature. Virology 350:433–442[CrossRef]
García-AndrésS.,
AccottoG. P.,
Navas-CastilloJ.,
MorionesE.2007a; Founder effect, plant host, and recombination shape the emergent population of begomoviruses that cause the tomato yellow leaf curl disease in the Mediterranean basin. Virology 359:302–312[CrossRef]
GussowD.,
ClacksonT.1989; Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res 17:4000[CrossRef]
HeathL.,
van der WaltE.,
VarsaniA.,
MartinD. P.2006; Recombination patterns in aphthoviruses mirror those found in other picornaviruses. J Virol 80:11827–11832[CrossRef]
Inoue-NagataA. K.,
AlbuquerqueL. C.,
RochaW. B.,
NagataT.2004; A simple method for cloning the complete begomovirus genome using the bacteriophage φ29 DNA polymerase. J Virol Methods 116:209–211[CrossRef]
IsnardM.,
GranierM.,
FrutosR.,
ReynaudB.,
PeterschmittM.1998; Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars. J Gen Virol 79:3091–3099
JeskeH.,
LutgemeierM.,
PreissW.2001; DNA forms indicate rolling circle and recombination-dependent replication of Abutilon mosaic virus. EMBO J 20:6158–6167[CrossRef]
KeightleyP. D.,
OttoS. P.2006; Interference among deleterious mutations favours sex and recombination in finite populations. Nature 443:89–92[CrossRef]
MartinD. P.,
van der WaltE.,
PosadaD.,
RybickiE. P.2005a; The evolutionary value of recombination is constrained by genome modularity. PLoS Genet 1:e51[CrossRef]
MeinschadC.,
WinnackerE. L.1980; Recombination in adenovirus. I. Analysis of recombinant viruses under non-selective conditions. J Gen Virol 48:219–224[CrossRef]
NdunguruJ.,
LeggJ. P.,
AvelingT. A.,
ThompsonG.,
FauquetC. M.2005; Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses. Virol J 2:21[CrossRef]
OdelbergS. J.,
WeissR. B.,
HataA.,
WhiteR.1995; Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I. Nucleic Acids Res 23:2049–2057[CrossRef]
OworB. E.,
MartinD. P.,
ShepherdD. N.,
EdemaR.,
MonjaneA. L.,
RybickiE. P.,
ThomsonJ. A.,
VarsaniA.2007a; Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant. J Gen Virol 88:3154–3165[CrossRef]
OworB. E.,
ShepherdD. N.,
TaylorN. J.,
EdemaR.,
MonjaneA. L.,
ThomsonJ. A.,
MartinD. P.,
VarsaniA.2007b; Successful application of FTA Classic Card technology and use of bacteriophage φ29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes. J Virol Methods 140:100–105[CrossRef]
PrasannaH. C.,
RaiM.2007; Detection and frequency of recombination in tomato-infecting begomoviruses of south and southeast Asia. Virol J 4:111[CrossRef]
SaundersK.,
LucyA.,
StanleyJ.1991; DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication. Nucleic Acids Res 19:2325–2330[CrossRef]
ShackeltonL. A.,
HoelzerK.,
ParrishC. R.,
HolmesE. C.2007; Comparative analysis reveals frequent recombination in the parvoviruses. J Gen Virol 88:3294–3301[CrossRef]
SharpP. M.,
RobertsonD. L.,
HahnB. H.1995; Cross-species transmission and recombination of ‘AIDS’ viruses. Philos Trans R Soc Lond B Biol Sci 349:41–47[CrossRef]
ShepherdD. N.,
MangwendeT.,
MartinD. P.,
BezuidenhoutM.,
ThomsonJ. A.,
RybickiE. P.2007; Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants. J Gen Virol 88:325–336[CrossRef]
ShepherdD. N.,
VarsaniA.,
WindramO. P.,
LefeuvreP.,
MonjaneA. L.,
OworB. E.,
MartinD. P.2008a; Novel Sugarcane streak and Sugarcane streak Réunion mastreviruses from southern Africa and La Réunion. Arch Virol 153:605–609[CrossRef]
ShepherdD. N.,
MartinD. P.,
LefeuvreP.,
MonjaneA. L.,
OworB. E.,
RybickiE. P.,
VarsaniA.2008b; A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. J Virol Methods 149:97–102[CrossRef]
StengerD. C.,
RevingtonG. N.,
StevensonM. C.,
BisaroD. M.1991; Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA. Proc Natl Acad Sci U S A 88:8029–8033[CrossRef]
TakeuchiY.,
HoriuchiT.,
KobayashiT.2003; Transcription-dependent recombination and the role of fork collision in yeast rDNA. Genes Dev 17:1497–1506[CrossRef]
VarsaniA.,
van der WaltE.,
HeathL.,
RybickiE. P.,
WilliamsonA. L.,
MartinD. P.2006; Evidence of ancient papillomavirus recombination. J Gen Virol 87:2527–2531[CrossRef]
VarsaniA.,
ShepherdD. N.,
MonjaneA. L.,
OworB. E.,
ErdmannJ. B.,
RybickiE. P.,
PeterschmittM.,
BriddonR. W.,
MarkhamP. G. & other authors (2008b). Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen. J Gen Virol 89:2063–2074[CrossRef]
WillmentJ. A.,
MartinD. P.,
RybickiE. P.2001; Analysis of the diversity of African streak mastreviruses using PCR-generated RFLPs and partial sequence data. J Virol Methods 93:75–87[CrossRef]
WillmentJ. A.,
MartinD. P.,
van der WaltE.,
RybickiE. P.2002; Biological and genomic sequence characterization of Maize streak virus isolates from wheat. Phytopathology 92:81–86[CrossRef]
WillmentJ. A.,
MartinD. P.,
PalmerK. E.,
SchnippenkoetterW. H.,
ShepherdD. N.,
RybickiE. P.2007; Identification of long intergenic region sequences involved in maize streak virus replication. J Gen Virol 88:1831–1841[CrossRef]
ZhouX.,
LiuY.,
RobinsonD. J.,
HarrisonB. D.1998; Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra. J Gen Virol 79:915–923