Appraising the apoptotic mimicry model and the role of phospholipids for poxvirus entry

Abstract

Entry of vaccinia virus (VACV) into cells occurs by fusion with the plasma membrane and via a low pH-dependent endosomal pathway, presumably involving unidentified cellular receptors. In addition to Ϸ25 viral proteins, the membrane of VACV mature virions contains several phospholipids including phosphatidylserine (PS). A recent model posits that PS flags virions as apoptotic debris to activate a common cellular uptake pathway to gain cell entry, perhaps through an interaction with a PS-specific cell surface receptor. To evaluate the apoptotic mimicry model, we reconstituted the membrane of detergent-extracted virions with several different phospholipids. Although the ability of the L-stereoisomer of PS to reconstitute infectivity was confirmed, the nonbiologically relevant D-stereoisomer of PS, and phosphatidylglycerol, which are not normally present in the virion membrane, functioned as well. Regardless of which phospholipid reconstituted infectivity, virus entry was inhibited by a neutralizing monoclonal antibody to a virion surface protein and by the drugs blebbistatin and bafilomycin A1, suggesting that in each case virus uptake was specific and occurred by a similar mechanism involving macropinocytosis and a low-pH endocytic pathway. Lipid-reconstituted and nonreconstituted, membrane-extracted virions were equally capable of binding to cells. However, the physical association of phospholipids with virus particles during membrane reconstitution correlated directly with rescue of particle infectivity and cell entry capability. Our results support a role for PS in poxvirus entry, but demonstrate that other phospholipids, not known to signal uptake of apoptotic debris, can function similarly.blebbing ͉ endocytosis ͉ macropinocytosis ͉ phosphatidylserine ͉ vaccinia virus

Vaccinia Virus L2 Protein Associates with the Endoplasmic Reticulum near the Growing Edge of Crescent Precursors of Immature Virions and Stabilizes a Subset of Viral Membrane Proteins

“…In our previous studies with the conditional lethal inducible L2 mutant vL2Ri, we noted that processing of the A17 membrane protein and the abundant core proteins were reduced, consistent with the early block in virion assembly demonstrated by electron microscopy (25). However, the overall […”

“…The recombinants vL2Ri (27), vF9Li (7), and vA17Li (56) were grown in the presence of 25 to 100 M IPTG (isopropyl-␤-Dthiogalactopyranoside). MVs were purified from cell lysates by sedimentation twice through 36% sucrose cushions, once in a 25 to 40% sucrose gradient as described previously (15) and once in a CsCl gradient (25).…”

Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry

“…A key trigger was in both cases the exposed PS in the viral envelope, the only lipid in the virus membrane specifically required for infectivity (5)(6)(7). Although the cellular PS-sensors involved remain unidentified, PS-receptors have been described mainly in macrophages as components of the machinery needed for endocytosis of apoptotic bodies.…”

The Tyro3 Receptor Kinase Axl Enhances Macropinocytosis of Zaire Ebolavirus

“…The amiloride analog EIPA inhibits the Na ϩ /H ϩ exchanger that blocks the formation of macropinosomes during macropinocytosis (50). Blebbistatin (BLB) has been shown to inhibit the formation of myosin II-dependent processes and has been used extensively as an inhibitor of macropinocytosis pathways (38,44,50,64). Cytochalasin B (CYTO-B) inhibits microfilament formation as well as actin polymerization (54).…”