Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

Abstract

The family Arenaviridae includes a number of highly pathogenic viruses that are responsible for acute hemorrhagic fevers in humans. Genetic diversity among arenavirus species in their respective rodent hosts supports the continued emergence of new pathogens. In the absence of available vaccines or therapeutic agents, the hemorrhagic fever arenaviruses remain a serious public health and biodefense concern. Arenaviruses are enveloped virions that assemble and bud from the plasma membrane. In this study, we have characterized the microdomain organization of the virus envelope glycoprotein (GPC) on the cell surface by using immunogold electron microscopy. We find that Junín virus (JUNV) GPC clusters into discrete microdomains of 120 to 160 nm in diameter and that this property of GPC is independent of its myristoylation and of coexpression with the virus matrix protein Z. In cells infected with the Candid#1 strain of JUNV, and in purified Candid#1 virions, these GPC microdomains are soluble in cold Triton X-100 detergent and are thus distinct from conventional lipid rafts, which are utilized by numerous other viruses for assembly. Virion morphogenesis ultimately requires colocalization of viral components, yet our dual-label immunogold staining studies failed to reveal a spatial association of Z with GPC microdomains. This observation may reflect either rapid Z-dependent budding of virus-like particles upon coassociation or a requirement for additional viral components in the assembly process. Together, these results provide new insight into the molecular basis for arenavirus morphogenesis.

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

“…Further observations of VSV glycoprotein and VSV matrix protein revealed different locations of the proteins at the plasma membrane, also indicating that the two proteins do not share affinities to certain lipids (77). A similar result was observed using electron microscopy analysis of the plasma membrane of cells transfected with plasmids encoding the Z protein and GP-C of the arenavirus Junin virus (1). Currently, participation of the glycoprotein and matrix protein of arenaviruses in lipid selection of the viral envelope has not been investigated.…”

“…For the New World arenavirus Junin virus, it was recently shown that assembly of GP occurs in detergent-soluble membrane microdomains (1). However, given the biological variations among New World and Old World arenaviruses in terms of receptor usage and mechanisms of cell entry, it is of interest whether these viruses also differ with respect to their assembly platform at the plasma membrane.…”

“…Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60,72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12,38,68).…”

Cis- and cell-type-dependent trans-requirements for Lassa virus-like particle production

“…2) indicate that these proteins are localized mainly at non-lipid rafts (detergentsoluble membrane areas). Considering these results, it is possible that once LASV Z and GPC reach the PM, they localize at non-lipid rafts but relocalize to the cholesterolrich domains (lipid rafts) just before budding and incorporate cholesterol into the virion, as proposed for JUNV (Agnihothram et al, 2009).…”

Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis

“…This so-called myristoyl switch is typically triggered by ligand binding and promotes a range of biologically significant changes in protein activity, protein-protein interactions, and membrane localization (26,27). In ectopically expressed GPC, the G2A mutation that abolishes myristate addition to SSP has been shown to reduce cell-cell fusion activity to ϳ20% of the wild-type level, without affecting GPC biosynthesis and trafficking (18,28) or its accumulation into discrete detergent-soluble membrane microdomains on the cell surface (29). The molecular basis for this fusion deficiency is unclear, and it is unknown whether GPC is similarly impacted when incorporated into virions.…”