Investigating the testicular tropism of Zika virus using 2D and 3D human tissue culture systems

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus traditionally associated with mild febrile illness. However, following the 2015–2016 epidemic in the Americas, new severe manifestations of ZIKV disease emerged, including microcephaly in neonates and Guillain-Barre syndrome in adults. Sexual transmission of ZIKV, which is atypical for flaviviruses, also became a common occurrence, predominantly male-to-female, during the epidemic. Cohort and case studies have since shown that ZIKV can be shed in semen regardless of symptoms for several months to more than a year following infection. This indicates a potentially long infectious period for the virus and highlights a risk of disease spread to non-endemic regions. Furthermore, prolonged seminal shedding of ZIKV in humans has been associated with reduction in motile sperm and altered serum levels of testosterone, providing evidence that ZIKV likely infects the testes and impacts testicular function. However, due to the lack of relevant small animal models, little is known regarding ZIKV tropism and infection in the testes, including the cell targets, immune responses, and pathophysiology. These gaps ultimately hamper efforts to properly assess the risks of ZIKV persistence for male reproductive health and also hinders the development of appropriate countermeasures. To begin to address these gaps, here we investigate various aspects of testicular ZIKV tropism and infection using different in vitro systems, including human 2D testis cell cultures and recently established 3D multicellular human testicular organoids (HTO). The testis is composed of two compartments, the interstitium and seminiferous tubules. Here, we show that seminiferous tubule cells (STC), including peritubular myoid cells, spermatogonial stem cells, and Sertoli cells (SC), were all susceptible to ZIKV; however, SC were clearly the most permissive to infection. In contrast, Leydig cells (LC), the major cell type of the interstitium that produces testosterone, were highly resistant to ZIKV. Basal protein levels of the TAM (Tyro3, Axl, Mer) receptor Axl and basal transcript levels of antiviral effectors correlated with the differential permissibility of SC and LC to ZIKV infection. We further document that HTO supported productive ZIKV infection, resulting in reduced HTO viability and function. Temporal transcriptomics analysis of infected SC showed that the most prominent response to ZIKV at the early stage of infection was the suppression of genes involved in cell growth and proliferation, while peak virus replication was associated with the induction of genes involved in innate antiviral defense. Similarly, our proteomics analysis demonstrated that ZIKV infection in SC and STC predominantly impacted pathways and functional networks associated with innate antiviral defense. Increased levels of the interferon (IFN)-I/III-stimulated protein MX1 at the later time points of infection coincided with diminished propagation of ZIKV in SC, and silencing of MX1 and the IFN-stimulated gene (ISG) IFIT1 enhanced peak ZIKV replication in SC. Although IFN-I signaling was found to restrict ZIKV replication in SC, further analysis demonstrated that SC exhibited delayed/dampened IFN response following ZIKV infection. Moreover, neutralization of Axl receptor and its ligand Gas6 strongly attenuated ZIKV entry in SC. However, inhibition of Axl kinase did not affect ZIKV entry but instead led to decreased protein levels of suppressor of cytokine signaling (SOCS)1 and SOCS3, increased expression of ISGs, and reduced ZIKV replication. Similarly, treatment with an Axl kinase inhibitor attenuated ZIKV replication and increased ISG expression in HTO. Collectively, our findings provide new insights into the testicular tropism of ZIKV. The data implicate SC as the principal cell target of robust ZIKV infection in the testes and demonstrate the utility of HTO for studying various aspects of testicular ZIKV infection and pathogenesis in lieu of animal models. Moreover, this study highlights the IFN-I/III response as a driver of the antiviral state that eventually limits ZIKV propagation in SC. Importantly, the data also suggest that delayed/dampened antiviral defense in SC may contribute to the ability of ZIKV to establish persistence in the testes. Furthermore, this study defines a novel role of Axl in modulating ZIKV infection in SC and as a determinant of ZIKV tropism in the testes.