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

Abstract

(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 co-flotation 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 Ca from the assay buffer or mutation of RuV envelope E1 protein Ca-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various non-lymphoid adherent cell lines independently of extracellular Ca or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca-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 Ca-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca-dependent and the other Ca-independent. Ca-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 Ca-independent RuV binding is an important next step in understanding the pathology of RuV infection. Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Despite effective rubella vaccines being available, rubella outbreaks still occur in many countries. We studied the entry mechanism of (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca at neutral pH. This Ca-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 Ca-independent manner. An unidentified RuV receptor(s) is involved in this Ca-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Natural ligand-nonmimetic inhibitors of the lipid-transfer protein CERT

“…Beyond its primary function to deliver ceramide from the ER to the Golgi apparatus, CERT participates in various biological events, including polyploid cancer cell death 32 , EGF receptor signaling 33 , lipotoxicity and glucolipotoxicity in islet β-cells 34,35 , muscle insulin signaling 36 , stress-induced Golgi disassembly 37 , protein secretions 38 , phosphoinositide turnover at the trans Golgi network 39 , cytotoxic autophagy 40 , and senescence 41 . Additionally, pharmacological or genetic inhibition of CERT negatively affects the proliferation of several types of intracellular pathogens [42][43][44][45][46] . Moreover, a point mutation causing the loss of repressive phosphorylation in the human CERT gene results in a hereditary mental development disorder with an autosomal dominant inheritance 11 .…”

Depletion of Host and Viral Sphingomyelin Impairs Influenza Virus Infection

“…It was also reported that alphaviruses require membrane sphingolipids, specifically SM, as a cofactor for virus fusion (Nieva et al, 1994). Moreover, Rubella virus was shown to directly bind to SM and cholesterol/SM-enriched microdomains (Otsuki et al, 2018). Lipidomic analyses have shown an increase in the SM content of IAV-infected cells (Tanner et al, 2014).…”

Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication

“…In RUBV infection experiments, recombinant RUBV rHS-p150/AG1, which expresses p150 fused with the humanized monomeric Azami green 1 (AG1) fluorescent protein, was used (21,22). In SINV infection experiments, the isolated MM2215 virus strain was used (23,24). The infected cells were cultured with various concentrations of 17-allylamino-17-desmethoxygeldanamycin , an inhibitor of HSP90 activity (25).…”

“…The recombinant virus rHS-p150/3FLAG, expressing p150 tagged with the 3FLAG epitope, was recovered from the genomic infectious cDNA clone pHS-p150/ 3FLAG. The MM2215 strain of SINV has been reported previously (23,24). RUBV and SINV were propagated in BHK cells.…”