Designed Host Cells Combined

Yet further increased complexity is realized in assays that rely on mixtures of different designed cells or cell types [83]. Although these assays are complicated, they yield insights into molecular mechanisms at the cell membrane, such as fusion events that are required for enveloped viruses to enter the target cell. Interactions at lipid bilayers with trans-membrane or integral proteins are difficult to simulate without cellular in-vitro systems. If the fusion event can be quantified, a screen for drugs that specifically target this very first step of infection is possible.

As mentioned above, the compound ALX 40-4C was rationally designed to interfere with transcription, but subsequently shown to inhibit fusion of the viral envelope with the cell membrane. The interference of ALX 40-4C with membrane fusion was performed by first generating two populations of cells: quail fibrosarcoma QT6-C5 target cells were transiently co-transfected with cellular receptors (CD4 and co-receptors) and a luciferase expression cassette under control of the bacteriophage T7 promoter. HeLa effector cells were infected with vaccinia virus vectors expressing HIV envelope protein and T7 polymerase. The two cell populations were mixed in the presence or absence of inhibitor, and the luciferase signal from cell lysates was assayed after 8-10 h [66]. In the absence of inhibitor, the viral envelope protein in the plasma membrane of effector cells and the transiently expressed receptor on the surface of target cells interacted, leading to activation of the viral fusion domain and thus formation of multinucleated syncitia. The cytoplasm of the syncitia then contained both T7-dependent reporter construct and cognate T7 polymerase, thereby allowing expression of the reporter and thus quantification of fusion activity.

A set-up that facilitates the fusion assay and, at the same time, combines assays for inhibitors of viral entry and transactivation in a single screen [84, 85] has been developed using a mixture of designed cells with cells chronically infected with HIV (a chronic infection, as opposed to latent infection, is characterized by a continuous release of virus). The indicator cells are adherent HeLa cells stably equipped with CD4 receptor and LTR-driven reporter, in this case lacZ. The chronically infected cells express viral envelope protein in the plasma membrane, but have down-regulated levels of CD4. If the indicator and the effector cells are co-cultivated, the interaction of CD4 on the HeLa derivatives and envelope protein on the producers cause fusion of the cell membranes. This leads to formation of the syncitia that can be assayed by microscopic examination. The transfer of Tat protein from the producer cells furthermore activates the reporter cassette in the indicator cells, and this can be visualized histologically or quantified by using spectroscopic galactosidase dyes. In the presence of the test substance, three possible readouts can be distinguished (Fig. 7.4): blue syncitia if the drug is without

Fig. 7.4 Fusion-induced gene stimulation (FIGS) assay. The SX22-1 indicator cells stably carry a TAT transactivator-responsive lacZ-cassette and express recombinant CD4 receptor molecule for HIV. The viral envelope protein (Env) is displayed in the membrane of the chronically infected HUT effector cells. (a) When the two cell types are mixed, interaction of CD4 and Env causes fusion of indicator and effector cells. This leads to formation of syncitia and transfer of Tat from the

Fig. 7.4 Fusion-induced gene stimulation (FIGS) assay. The SX22-1 indicator cells stably carry a TAT transactivator-responsive lacZ-cassette and express recombinant CD4 receptor molecule for HIV. The viral envelope protein (Env) is displayed in the membrane of the chronically infected HUT effector cells. (a) When the two cell types are mixed, interaction of CD4 and Env causes fusion of indicator and effector cells. This leads to formation of syncitia and transfer of Tat from the effector to the indicator cell, thus activating lacZ expression that can be visualized or quantified. (b) If a drug interferes with cell fusion (or virus entry), no syncitia are formed and, as Tat is not transferred, the cells also do not express lacZ. (c) If a drug interferes with Tat activity, fusion (and therefore syncitia formation) is not suppressed but reporter gene expression is not transactivated, leading to formation of unstained syncitia.

effects; white (lacZ-negative) syncitia if the drug interferes with transactivation; and conventional unstained monolayer of cells if the drug prevents membrane fusion and virus entry.

Lessons learned from HIV screens can be transferred to other fields of infectious diseases. For example, the recent development of replicon systems for HCV has led to in-vitro drug screens [86] against this serious chronic disease that affects 170 million people worldwide. The HCV replicon is a subviral RNA molecule maintained within the host cell without extracellular states. The variations for cell-based screens include systems with specially selected cells that support robust replicon expression, and replicons that carry and express reporter genes to facilitate quantification of interfering drugs in high-throughput systems. Highly illustrative with respect to this chapter, HCV replicons have been created that release HIV Tat to activate a stably inserted LTR-reporter cassette in HCV-permissive Huh-7 cells [87]. The in-vitro screens have already yielded a number of promising compounds directed against viral proteases and polymerases. In addition, the in-vitro study of the complete life cycle of HCV has finally become accessible, with recent developments suggesting that cell culture-based screens against viral entry and uncoating or morphogenesis also should be possible in the near future [88]. Such studies will, hopefully, be guided and accelerated by the experience with HIV that dates back to the 1980s.

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