We have previously shown that the G protein ofvesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracyclinedependent and can be modulated by 18-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.Retroviral vectors derived from Moloney murine leukemia virus have been used extensively to transduce genes into mammalian cells (1, 2). However, problems such as the inability to infect quiescent cells, low virus titers, and relatively poor infectivity in human cells severely limit the application of retroviral vectors in gene transfer. To overcome some of these problems, we have generated retroviral vectors pseudotyped with the G protein of vesicular stomatitis virus (VSV-G) (3-5). We have shown that the pseudotyped retroviral vectors have an expanded host range and are able to infect many cell types with a greater efficiency than traditional amphotropic retroviral vectors (3-6). Moreover, the pseudotyped virus can be concentrated by ultracentrifugation to titers greater than 109 colony forming units/ml (4). These unique properties of the VSV-G pseudotyped virus not only extend the use of retroviral vectors for genetic studies in previously inaccessible species, but also facilitate more efficient preclinical and clinical studies of the potential for human gene therapy.However, the generation of stable packaging cell lines for pseudotyped retroviral vectors has been difficult because of the toxicity of VSV-G in most mammalian cells (4, 5). To overcome this problem, we have modified the tetracyclineinducible system (7) to regulate the expression of VSV-G. In that system, a chimeric transcription factor, tTA, was produced by combining the repressor of Escherichia coli tetracyclineresistance operon (tetR) and the activation domain of virion protein 16 (VP16) of herpes simplex virus (HSV). This chimeric transactivator can regulate gene expression from a minimal promoter linked to multiple copies of tetO, the binding site for the tetracycline repressor. In the absence of tetracycline, tTA binds the tetO site and activates transcription from the tetO-containing promoter. In the presence of tetracycline, interaction of tTA with the tetO is prevented by its binding to tetracycline, thereby prevents transcription.While the expression of several genes has been reported to be inducible using this system (7-11), establishment of cell lines stably expressing tTA has been difficult, presumably because the activation domain of VP16 is capable of squelching general cellular transcription when expressed in l...
In the absence of envelope gene expression, retrovirus packaging cell lines expressing Moloney murine leukemia virus (MLV)gag and pol genes produce large amounts of noninfectious virus-like particles that contain reverse transcriptase, processed Gag protein, and viral RNA (gag-pol RNA particles). We demonstrate that these particles can be made infectious in an in vitro, cell-free system by the addition of a surrogate envelope protein, the G spike glycoprotein of vesicular stomatitis virus (VSV-G). The appearance of infectivity is accompanied by physical association of the G protein with the immature, noninfectious virus particles. Similarly, exposure in vitro of wild-type VSV-G to a fusion-defective pseudotyped virus containing a mutant VSV-G markedly increases the infectivity of the virus to titers similar to those of conventional VSV-G pseudotyped viruses. Furthermore, similar treatment of an amphotropic murine leukemia virus significantly allows infection of BHK cells not otherwise susceptible to infection with native amphotropic virus. The partially cell-free virus maturation system reported here should be useful for studies aimed at the preparation of tissue-targeted retrovirus vectors and will also aid in studies of nucleocapsid-envelope interactions during budding and of virus assembly and virus-receptor interactions during virus uptake into infected cells. It may also represent a potentially useful step toward the eventual development of a completely cell-free retrovirus assembly system.
Tlwo regions of the hepatitis B virus (HBV) genome have been shown to display properties of a transcriptional enhancer. Enhancer 1 is active in most hepatoma lines examined as well as in some non-hepatocyte-derived cell lines. In contrast, enhancer 2 activity is strictly liver specific. In this study, we show that adenovirus EIA expression in the highly differentiated human hepatoma line Huh6 strongly inhibits HBV enhancer 2-stimulated transcription while having no effect on HBV enhancer 1 activity. A sequence motif in HBV enhancer 2 which is essential for its enhancer function is the target for ElA-mediated repression. The repression of HBV enhancer 2 activity is mediated through the N-terminal region of the E1A proteins known to bind a 300-kDa cellular protein. Our results suggest that HBV enhancer function may be modulated by a cellular mechanism similar to ElA-mediated transcriptional repression.
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