Rev-dependent lentiviral expression vector.
BACKGROUND
HIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells.


RESULTS
We have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency.


CONCLUSIONS
As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.
Wu
Yuntao
Y
Section on Molecular Virology, Laboratory of Cellular and Molecular Regulation, NIMH, Bethesda, MD 20892-4483, USA. ywu8@gmu.edu
Beddall
Margaret H
MH
Marsh
Jon W
JW
D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression; D015694:Gene Products, rev / Q000378:metabolism*; D017930:Genes, Reporter; D015752:Genes, env / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006678:HIV / Q000235:genetics* / Q000502:physiology*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D054321:rev Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015694:Gene Products, rev; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D054321:rev Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins