A mechanistic study of how hepatic cytochrome P450 3A4 is regulated during infectious and non-infectious conditions
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It has been established that adenovirus infection alters the expression and function of hepatic cytochrome P450 3A (CYP3A) for 14 days in several animal models. To determine the mechanism behind adenovirus-mediated alterations in CYP3A4 and if this effect was relevant to humans, we characterized a human hepatocyte cell line, HC-04, as an in vitro model to study virus-mediated changes in human CYP3A4. Similar to in vivo observations, infection with a first-generation recombinant adenovirus (AdlacZ) significantly suppressed CYP3A4 catalytic activity in an isoform-specific manner in this cell line. HC-04 cells were subsequently used to determine how vaccines to be used in pandemic outbreaks impact CYP3A4 expression and function. Infection with a recombinant adenovirus-based Ebola vaccine or influenza A virus subtypes H1N1 and H3N2 significantly suppressed human hepatic CYP3A4 catalytic activity by 34%, 91%, and 90%, respectively. This effect is important to understand as these vaccines are given to people of all ages. Mechanistic study of adenovirus infection in HC-04 cells indicated that engagement of integrin receptors is key in the initiation of processes responsible for changes in CYP3A4 during infection. Mice infected with AdlacZ experienced a 70% reduction in CYP3A activity 24 hours after infection. While infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the viral capsid (AdΔRGD) did not alter CYP3A activity at the same timepoint. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were similar to uninfected control mice. Silencing of the integrin β-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of the α-subunit did not. This led us to believe that interactions with the -tail of the integrin receptor is important for virus-mediated suppression of CYP3A4. Talin-1, a cytoplasmic protein that specifically binds to the -tail of integrins therefore became the focus of remaining studies. Knockdown of talin-1 increased CYP3A4 catalytic activity by 190%. Genes associated with post-translational modifications (PTMs) were downregulated in talin-silenced samples and analysis during viral infection revealed a significant increase in PTMs of CYP3A4. These initial experiments indicate a role of talin-1 and PTMs in the regulation of CYP3A4, however further experiments are needed to confirm these findings. Understanding changes in CYP3A4 during wild-type infection and immunization is clinically important, especially for low metabolizers. In this population, suppression of CYP3A4 could lead to accumulation of drugs that normally would be metabolized and cleared via this pathway, leading to serious therapeutic failure or adverse effects.