Construction and expression of a Rev-dependent TNF-R1 expressing HIV-infected-cell injurious vectors.
BACKGROUND
Rev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE).


METHODS
Molecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope.


RESULTS
The new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours.


CONCLUSIONS
Rev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.
Shi
Wei-min
WM
Department of Dermatology and Venereology, Tongji Hospital, Tongji University, Shanghai 200065, China. shiweimin1234@medmail.com.cn
Baylis
Dean
D
Purcell
Damian
D
Cameron
Paul U
PU
D016915:AIDS Vaccines / Q000276:immunology*; D017209:Apoptosis; D019470:Biolistics; D045744:Cell Line, Tumor; D015694:Gene Products, rev / Q000502:physiology*; D015752:Genes, env / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D010957:Plasmids; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics*
D016915:AIDS Vaccines; D015694:Gene Products, rev; D047888:Receptors, Tumor Necrosis Factor, Type I