Fatty Acid-Depleted Albumin Induces the Formation of Echovirus A Particles

Abstract

Picornavirus infection requires virus uncoating, associated with the production of 135S "A" particles and 80S empty particles from 160S mature virions, to release the RNA genome into the cell cytoplasm. Normal albumin inhibits this process. We now show that when depleted of fatty acids, albumin induces the formation of echovirus A particles.Echoviruses (EVs), members of the Enterovirus genus of the family Picornaviridae, are small positive-strand RNA viruses with icosahedral symmetry which produce a broad spectrum of diseases in humans (8). Productive cell infection requires the uncoating of the virus particle and release of the RNA genome into the cell cytoplasm. The conversion of the 160S mature virions into 135S A and 80S empty particles (reviewed in reference 14) is considered indicative of this process. Poliovirus A particle formation can be induced by soluble poliovirus receptor (6, 20), whereas soluble decay-accelerating factor, a receptor for a number of enteroviruses including EV12 (17), does not induce EV A particles (12). Moreover, conversion of poliovirus to A particles is rapid compared with the conversion of EV (12). Both uncoating and the thermal stability of virions are thought to be regulated by the presence of "pocket factors" in the hydrophobic pocket at the base of the canyon floor. Known pocket factors are fatty acid (FA) or FA based; poliovirus uses sphingosine (14), coxsackievirus B3 and EV1 both use palmitate (3, 11), and bovine enterovirus uses myristate (15). Enterovirus uncoating can be inhibited by pocket factor mimetics like WIN compounds (10,14) and by the pocket factor itself (5). The uncoating of EV, but not poliovirus, can also be blocked by normal albumin (18); since this protein contains FAs (7), we considered the possibility that FAs were responsible for this block. However, FA-depleted albumin also blocks EV infection (18). We have now investigated the block on EV infection caused by FA-depleted albumin by using wildtype (WT) EV12 and three thermolabile rhodanine-resistant EV12 mutants (1,2,8).Normal bovine serum albumin (A-7638; Sigma) and FAdepleted albumin (A-0281; Sigma) were solubilized in serumfree Dulbecco modified Eagle medium (DMEM) (final pH 7.4). EV12 and the rhodanine-resistant mutants numbered 9, 17, and 20 were incubated for 1 h at 37°C with normal or FA-depleted albumin (2%, wt/vol), or rhodanine (200 g/ml). Virus was then diluted in serum-free DMEM and adsorbed to 24-well plates of rhabdomyosarcoma (RD) cells at 95% confluency for 30 min. Cells were incubated in serum-free DMEM, and at 7 h postinfection, cells were assayed for infected cells by using an immunofocal assay (18). Preincubation of WT EV12 or mutants 17 or 20 with normal albumin and rhodanine did not affect infectivity, whereas infection by mutant 9 was enhanced by 180% (Fig. 1). In contrast, infectivity of all the viruses was reduced after preincubation with FA-depleted albumin; WT EV12 was decreased by 50%, and infectivity of the more thermolabile rhodanine mutants (8) was reduced by 70% (mutant ...

Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation

“…3F), and the approximately 1:1 ratio of albumin and fatty acids observed in serum, it seems probable that under physiological conditions, the albumin present outside the cells starts the uncoating process. The ability of faf-BSA to induce echovirus A-particle formation was previously shown using a radioactive gradient; however, the nature of this process was not studied further (21). Our results here explain the mechanism by which faf-BSA induces uncoating in enteroviruses, such as echoviruses and coxsackieviruses.…”

“…They have also been found to exist in purified virus preparations (20). One study showed that treatment with fatty acid-free bovine serum albumin (BSA) converts echovirus 12 into A-particles, but the mechanism of action was not investigated further (21). In addition, there are only few studies reporting what effects other physiological factors, such as changes in ionic conditions, may have on the virus particle (21)(22)(23)(24)(25).…”

“…One study showed that treatment with fatty acid-free bovine serum albumin (BSA) converts echovirus 12 into A-particles, but the mechanism of action was not investigated further (21). In addition, there are only few studies reporting what effects other physiological factors, such as changes in ionic conditions, may have on the virus particle (21)(22)(23)(24)(25). X-ray crystallography as well as cryo-electron microscopy (cryoEM) and single particle reconstruction have been used to gain structural information on picornavirus particles at different stages before genome release (20,(26)(27)(28)(29)(30)(31)(32)(33).…”

Characterization of Early Steps in the Poliovirus Infection Process: Receptor-Decorated Liposomes Induce Conversion of the Virus to Membrane-Anchored Entry-Intermediate Particles

“…In the absence of these components, virus conversion was able to proceed directly to the 80S particle, independent of both temperature and receptor (data not shown), perhaps triggered by a membrane interaction resulting from transient externalization of VP1. Previous studies have documented the ability of BSA (or fatty acid components present in preparations of BSA) or myristate to stabilize picornavirus particles (11,33,34). In the current study, BSA may also have stabilized the virus, leading to reduced virus breathing and therefore reduced nonreceptor-mediated membrane interaction.…”

Albumin Enhances the Rate at Which Coxsackievirus B3 Strain 28 Converts to A-Particles

“…Enterovirus breathing is believed to require equilibrium between occupied and empty pockets; i.e., pocket factor must be displaced for the capsid conformation to open (6,7). Albumin is the principal fatty acid-binding protein in serum, and defatted albumin has been shown to promote an echovirus conversion to A-particles and to increase permeability to small molecules (33,35). The measured fatty acid content of the BSA purified from FBS was substantially less than saturating, so the BSA in these experiments has ample reserve capacity to bind fatty acids (36).…”