Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry

Abstract

Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca²⁺ from the assay buffer or mutation of RuV envelope E1 protein Ca²⁺-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca²⁺ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca²⁺-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca²⁺-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca²⁺ dependent and the other is Ca²⁺ independent. Ca²⁺-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca²⁺-independent RuV binding is an important next step in understanding the pathology of RuV infection.IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca²⁺ at neutral pH. This Ca²⁺-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca²⁺-independent manner. An unidentified RuV receptor(s) is involved in this Ca²⁺-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Keywords: cholesterol; lipid; rubella; sphingomyelin; virus entry.