Zika virus (ZIKV) is primarily transmitted to humans through the bite of an infected mosquito vector, but sexual transmission from men to women is possible. Alarmingly, ZIKV transmitted through a sexual route may pose a greater risk of fetal Zika syndrome when the infected subject is a pregnant woman. However, despite the high ZIKV load in the semen of infected men, rates of sexual ZIKV transmission from men to women is lower than expected based on viral load alone, suggesting that other factors may influence the ability of ZIKV to infect female genital cells.
The Vojtech and Hladik labs from the University of Washington Department of Obstetrics and Gynecology, along with the Fred Hutch Vaccine and Infectious Disease Division, have previously shown that seminal extracellular vesicles (SEV), lipid membrane-bound particles containing bioactive cargo, can influence sexually transmitted viral infection in female genital mucosa. In a recent publication in Frontiers in Microbiology, Ruofan Wang (Chan-Zuckerberg Biohub, formerly of the University of Washington) along with collaborators from the Vojtech and Hladik labs, investigated ZIKV transmission dynamics in female genital mucosa. They focused on a specific question: when ZIKV is sexually transmitted through semen, how do SEV influence infection dynamics?
Using previously established models of in vitro and ex-vivo vaginal epithelial cell culture, Wang and colleagues first demonstrated that ZIKV productively infected the vaginal mucosa and induced a strong antiviral immune response, measured by the production of inflammatory cytokines that are triggered through viral sensing pathways of the innate immune system. However, when ZIKV was combined with SEV in the cell culture system, ZIKV infection was reduced. When the cells were cultured with ZIKV and SEV for an initial period and then washed, ZIKV infection remained impaired, suggesting that SEV interfere with the initial steps of ZIKV binding and entry into host cells.
SEV contain proteins, nucleic acids, and lipids. To determine which SEV cargo components are responsible for inhibiting ZIKV cell entry, the authors employed various treatments to specifically disrupt each of the component types and repeated their initial experiments. Using heat to denature SEV proteins, they observed that denatured SEV inhibited ZIKV infection to the same degree as untreated SEV. To test the contribution of the lipid fraction of SEV, Wang and colleagues created nucleic acid-free liposomes of the same size and lipid content as SEV. They incubated these liposomes with ZIKV and vaginal epithelial cells and found that liposomes inhibited ZIKV infection at a similar rate to SEV, demonstrating that the lipid cargo of SEV is responsible for the disruption of cell entry. SEV lipids, like ZIKV, contain exposed phosphatidylserine (PS) which bind various host cell receptors and mediate one route of viral entry. A second route of viral entry is mediated by host receptor binding by viral glycoproteins. The authors hypothesized that SEV could inhibit ZIKV entry by blocking PS-binding receptors, which should not affect glycoprotein-mediated entry. However, when a cell line was transfected with either a PS-mediated or a glycoprotein-mediated host receptor, SEV inhibited ZIKV infection equally, demonstrating that a different mechanism was likely responsible for ZIKV inhibition. They then reasoned that SEV may directly interact with ZIKV, possibly by fusing with the virions and decreasing viral integrity. When ZIKV was treated with SEV or liposomes and later RNAse (which can access and destroy unprotected viral genomes, but not those of intact viruses), both SEV and liposomes caused a decrease in detectable ZIKV genome, suggesting that SEV lipids directly interact with ZIKV with virocidal effects.
By culturing vaginal epithelial cells with ZIKV both in the presence and absence of semen, Wang and colleagues demonstrated that although ZIKV can productively enter vaginal epithelial cells and cause a robust innate immune response, SEV can inhibit this entry. Although this finding supported their hypothesis that SEV modulate ZIKV infection in the genital mucosa, “the apparent mechanism of restriction of Zika virus by EVs in semen surprised us,” Dr. Vojtech said. “We predicted EVs would outcompete phosphatidylserine receptors on cells to prevent Zika binding. After numerous different types of experiments, it seems the mechanism is more complicated than that.”
These findings suggest that SEV may inhibit sexual transmission of other viruses, and that the contributions of semen on vaginal viral infections should be considered in the development of future therapeutics and vaccines. Going forward, Dr. Vojtech said that her lab is “excited to continue research into exactly how semen EVs impair viral infection and hope someday the mechanism might be harnessed for therapeutics."
This work was supported by the National Institutes of Health and the University of Washington Department of Obstetrics and Gynecology academic enrichment funds.
UW/Fred Hutch Cancer Consortium member Florian Hladik contributed to this work.
Wang R, Gornalusse GG, Kim Yeseul, Pandey U, Hladik F, Vojtech L. Potent Restriction of Sexual Zika Virus Infection by the Lipid Fraction of Extracellular Vesicles in Semen. Frontiers in Microbiology, 2020; 11: 574054.