Epstein-Barr Virus Lytic Replication Induces ACE2 Expression and Enhances SARS-CoV-2 Pseudotyped Virus Entry in Epithelial Cells

Understanding factors that affect the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is central to combatting coronavirus disease 2019 (COVID-19). The virus surface spike protein of SARS-CoV-2 mediates viral entry into cells by binding to the ACE2 receptor on epithelial cells and promoting fusion. We found that Epstein-Barr virus (EBV) induces ACE2 expression when it enters the lytic replicative cycle in epithelial cells. By using vesicular stomatitis virus (VSV) particles pseudotyped with the SARS-CoV-2 spike protein, we showed that lytic EBV replication enhances ACE2-dependent SARS-CoV-2 pseudovirus entry. We found that the ACE2 promoter contains response elements for Zta, an EBV transcriptional activator that is essential for EBV entry into the lytic cycle of replication. Zta preferentially acts on methylated promoters, allowing it to reactivate epigenetically silenced EBV promoters from latency. By using promoter assays, we showed that Zta directly activates methylated ACE2 promoters. Infection of normal oral keratinocytes with EBV leads to lytic replication in some of the infected cells, induces ACE2 expression, and enhances SARS-CoV-2 pseudovirus entry. These data suggest that subclinical EBV replication and lytic gene expression in epithelial cells, which is ubiquitous in the human population, may enhance the efficiency and extent of SARS-CoV-2 infection of epithelial cells by transcriptionally activating ACE2 and increasing its cell surface expression. IMPORTANCE SARS-CoV-2, the coronavirus responsible for COVID-19, has caused a pandemic leading to millions of infections and deaths worldwide. Identifying the factors governing susceptibility to SARS-CoV-2 is important in order to develop strategies to prevent SARS-CoV-2 infection. We show that Epstein-Barr virus, which infects and persists in >90% of adult humans, increases susceptibility of epithelial cells to infection by SARS-CoV-2. EBV, when it reactivates from latency or infects epithelial cells, increases expression of ACE2, the cellular receptor for SARS-CoV-2, enhancing infection by SARS-CoV-2. Inhibiting EBV replication with antivirals may therefore decrease susceptibility to SARS-CoV-2 infection.