ZIKV infection during pregnancy could result in a neonate with congenital Zika syndrome or other abnormalities. Therefore, developing an efficacious and low-priced vaccine for pregnant women must be given high priority in public health. Vaccines against ZIKV are in various stages of preclinical and clinical testing, with some having been in Phase I and II trials; however, not a single one has reached the population yet (Pattnaik et al. 2020). Besides, excluding pregnant women from vaccine clinical trials is still problematic; for those reasons, a treatment that is safe and accessible is one of the main goals of ZIKV infection studies. Here, we report the in vitro antiviral potential of metformin, an insulin-sensitizing and glucose-lowering drug, against ZIKV. We showed that metformin inhibits the ZIKV replication cycle in a dose-dependent manner and reverts the changes in cytoskeleton and lipid storage induced by ZIKV when it activates metabolic enzymes.
Metformin has been proven as a potential treatment against some infections with RNA viruses, such as severe acute respiratory syndrome coronavirus 2 or other flaviviruses, including DENV and HCV (Harris and Smith 2013; Chen et al. 2018; Samuel et al. 2021; Cheang et al. 2021), and also in DNA virus (Hoppe-Seyler et al. 2021; Zhang et al. 2022a). Our results demonstrate the capability of this drug to limit the viral infection in the first-trimester trophoblast cell line JEG3, at concentrations as low as 1 mM. Furthermore, the given drug doses did not have any significant cytotoxic effects on this cell line, similar to other trophoblast cell lines such as BeWo (Farfan-Morales et al. 2021a). This suggests a low risk of the drug in placental tissues since no teratogenic effects in the fetus or placental disturbance have been observed (Arshad et al. 2022).
According to our results, metformin treatment impacted viral replication at concentrations of 1–4 mM. Markedly, the reduction pattern was similar in all cases, decreasing the presence of viral replication components and expression at 50% average (1 mM) and getting lower than 25% and 10% (2.5 and 4 mM, respectively). Comparably, Morales et al. have demonstrated that metformin has a broad in vitro antiviral spectrum inhibiting ZIKV infection but more efficiently DENV and yellow fever virus in glioblastoma and hepatocyte cell models. Interestingly, they also observed that in the trophoblast cell line BeWo, which is less permissive to ZIKV infection than the JEG3 model, metformin showed a modest antiviral effect. Nonetheless, in immunodeficient mice, the drug had no effect on ZIKV infection; however, it ameliorated and reduced signs of disease in DENV infection (Farfan-Morales et al. 2021a).
Viruses are intracellular pathogens that perturb host cellular metabolism to for their replication and spread. Our results indicate a reduction in the phosphorylated form of AMPK (p-T172), suggesting an inhibition of the activity of this molecule (p-T172); in comparison with Mock cells. Interestingly, ZIKV-infected metformin-treated cells displayed a restoration of AMPK phosphorylation, regulating the effect of ZIKV infection. Singh and col. have confirmed this data. In endothelial cells infected with ZIKV, the phosphorylation in p-T172 of AMPK is reduced, and its activity is altered. The pharmacology activation of this enzyme by AICAR restricts ZIKV replication and potentiates antiviral pathways (Singh et al. 2020b). However, our results also show that lipogenic factors (SREBP2, HMGCoAR, and FAS), which drive the de novo synthesis of lipids such as cholesterol, are related to LDs, and membrane formation is activated or overexpressed. A closely related virus, DENV, increases HMGCR activity through AMPK inactivation, leading to higher cholesterol levels in the endoplasmic reticulum, which is necessary for replicative complexes (RCs). Moreover, metformin and lovastatin (HMGCR inhibitor) decline HMGCR activity and also alter the co-localization with HMGCoAR and cholesterol with no structural protein, suggesting a role for HMGCR and AMPK activity in the formation of flavivirus RCs (Soto-Acosta et al. 2017). Herein, ZIKV infection of placental tissue induces the accumulation of LDs. In this study, metformin treatment undermined LD formation, supporting the evidence that the drug regulates catabolism by reducing the availability of lipids necessary for the RC formation or lipotoxicity in placenta (Chen et al. 2020). The effect of metformin in JEG-3 cells may be related to the regulation of this lipogenic pathway and could directly be involved in the inhibition of ZIKV replication.
Additionally, our results related to transmission electronic microscopy evidenced that cytoskeletal effects during ZIKV infection, specifically IFs, are arranged in an aligned pattern. Concurrently, the metformin treatment disrupts this pattern. Vimentin is a part of IF, and during ZIKV infection, it suffers severe reorganization upon viral protein synthesis to form a perinuclear cage-like structure that concentrates RC. Therefore, vimentin-knockout cells markedly disturbed the integrity of RCs. These consequences resulted in fragmented subcellular dispersion of viral proteins and reduced replication and down-regulated genes involved in antiviral and inflammatory responses (Zhang et al. 2022b). Taking this into account, metformin treatment in gastric cancer cells (AGS) reduces the expression of vimentin and cell motility, migration, and invasion abilities of AGS cells (Valaee et al. 2021). Therefore, using this drug might modulate the RC complex during ZIKV infection; Morales and col. observed a decrease in the number of RCs in infected cells treated with metformin by transmission electronic microscopy. Furthermore, metformin disrupts the co-localization with NS4A -E ZIKV proteins, which are necessary to maintain RC (Farfan-Morales et al. 2021a).
Finally, it is relevant to mention an antiviral property that has been attributed to metformin; the pleiotropic properties of the drug suggest that it acts in multiple forms during infection in vitro. In combination, metformin and lamivudine enhanced the inhibitory effects of interferon-α2b on HB surface antigen expression and HBV replication (Xun et al. 2014). Similarly, in HCV-JFH1 replicon infection, metformin activated STAT-1 and STAT-2 phosphorylation in infected JFH-1 cells. Therefore, the activated IFN signaling inhibits the HCV replication by activating AMPK. After treatment with an AMPK inhibitor, the level of HCV core protein decreased by metformin can be rescued (Tsai et al. 2017). Future experiments to evaluate the metformin’s capability to stimulate immune response during ZIKV infection in the placenta would be considered.