Construction and applications of yellow fever virus replicons.
Subgenomic replicons of yellow fever virus (YFV) were constructed to allow expression of heterologous reporter genes in a replication-dependent manner. Expression of the antibiotic resistance gene neomycin phosphotransferase II (Neo) from one of these YFV replicons allowed selection of a stable population of cells (BHK-REP cells) in which the YFV replicon persistently replicated. BHK-REP cells were successfully used to trans-complement replication-defective YFV replicons harboring large internal deletions within either the NS1 or NS3 proteins. Although replicons with large deletions in either NS1 or NS3 were trans-complemented in BHK-REP, replicons that contained deletions of NS3 were trans-complemented at lower levels. In addition, replicons that retained the N-terminal protease domain of NS3 in cis were trans-complemented with higher efficiency than replicons in which both the protease and helicase domains of NS3 were deleted. To study packaging of YFV replicons, Sindbis replicons were constructed that expressed the YFV structural proteins in trans. Using these Sindbis replicons, both replication-competent and trans-complemented, replication-defective YFV replicons could be packaged into pseudo-infectious particles (PIPs). Although these results eliminate a potential role of either NS1 or full-length NS3 in cis for packaging and assembly of the flavivirus virion, they do not preclude the possibility that these proteins may act in trans during these processes.
Jones
Christopher T
CT
Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054, USA.
Patkar
Chinmay G
CG
Kuhn
Richard J
RJ
D002460:Cell Line; D005416:Flavivirus / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D012093:Replicon; D012845:Sindbis Virus / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology*; D014779:Virus Replication; D015005:Yellow fever virus / Q000235:genetics* / Q000502:physiology
D015678:Viral Structural Proteins; D049452:Green Fluorescent Proteins