Dissecting the Functional Domains of a Nonenveloped Virus Membrane Penetration Peptide

Abstract

Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the "gamma" peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.The presence of membrane lytic peptides in many nonenveloped viruses is well established (3, 16), but how these peptides are deployed from the virus capsid during host cell entry and disrupt membranes remains unclear. These peptides are typically generated by a postassembly proteolytic processing event (1, 11) and are exposed from a previously buried position during conformational alterations in the capsid triggered by host cell conditions (2, 18). Flock House virus (FHV), an insect nodavirus, contains a 4-kDa peptide called "gamma" (␥), which shares many of the characteristics of other nonenveloped virus lytic peptides (3). The FHV capsid is made from 180 copies of a single-coat protein (␣) enclosing a single-stranded bipartite RNA genome (9). Gamma is generated by the autocatalytic cleavage of ␣ during virus maturation (␣ 3 ␤ ϩ ␥) (15), remains localized in the capsid interior (9) with occasional externalization or "breathing" (6), and is exposed under low-pH conditions in the endosomes during entry (Odegard et al., submitted for publication). Covalently independent gamma is necessary for virus infection, since maturation-defective FHV (D75N/N363T FHV), which does not undergo the autocleavage of ␣, is not infectious (15,17). The N-terminal ϳ21 residues of gamma (corresponding to residues 364 to 384 of ␣) constitute an amphipathic helix which can disrupt membranes in vitro when synthetically produced (4, 5) and is recognized as the host membrane-interacting region of FHV during entry. The hydrophobic, ϳ23-residue-long C terminus of gamma, especially certain phenylalanine residues (at positions 402, 405, and 407), is responsible for specifically packaging viral RNA into capsids during assembly (14).It was recently demonstrated that a supply of full-length gamma from noninfectious virus-like particles (VLPs) of FHV (13) during entry can restore infectivity to maturation-defective FHV (17), suggesting that gamma can function in trans to mediate access into host cells. This trans-complementation assay (17) was utilized to provide a quantitative readout of the effect of gamma mutations specifically on virus entry and to thus assess the region(s) of gamma required during virus entry. To determine the minimal sequence of gamma required for trans-complementation, FHV VLPs were produced that included the first 384, 390, or 395 amino acids of capsid protein ...

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

“…Previous studies have demonstrated that mature wild-type VLPs can rescue the infectivity of noninfectious, maturation-defective FHV (FHV MD ), and the extent of rescue can be quantified by specifically labeling the progeny virus produced with 35 S (40). The presence of cleaved, full-length gamma peptide in VLPs is essential for rescue (5,40). The calcium-site-mutated VLPs, although they contained full-length cleaved gamma peptides, could not rescue the infectivity of FHV MD (Fig.…”

“…Membrane disruption by FHV particles is due to the activity of the gamma peptide, which is primarily sequestered inside the capsid at a neutral pH, with only occasional externalization (7,26). Gamma becomes fully exposed in the low-pH environment inside early endosomes, which correlates with increased membrane disruption demonstrated by wildtype FHV at pH 6.0 in vitro (5,26). The pattern of membrane disruption by FHV CM3 VLPs indicates that the exposure of gamma in this particle is no longer controlled by pH conditions.…”

Low Endocytic pH and Capsid Protein Autocleavage Are Critical Components of Flock House Virus Cell Entry

“…Therefore, it is possible that the viral RNA remains associated with the ␥ peptides as they are released from the capsid interior, and the RNA travels along with the ␥ peptides to the site of membrane penetration or into the cytoplasm. Studies are currently under way to address the role of the RNA binding region of ␥ in membrane penetration (5).…”

Dissecting Quasi-Equivalence in Nonenveloped Viruses: Membrane Disruption Is Promoted by Lytic Peptides Released from Subunit Pentamers, Not Hexamers

“…The role of the lytic peptide, and associated infectivity, in Flock House virus (FHV), a nonenveloped, Tϭ3 quasi-equivalent, small-subunit RNA (ssRNA) insect virus, was investigated with extensive mutagenesis in vivo employing a novel protocol in which the lytic peptide was provided in trans with a virus-like particle (VLP) (32). There was a striking correlation between mutants that show reduced dye release in vitro and reduced infectivity in vivo, thus confirming the biological relevance of the liposome assay (2).…”

“…A common feature of many nonenveloped animal viruses is a capsid-associated lytic peptide that is activated by a chemical cue and assumed to facilitate membrane rupture for genome or particle delivery to the cytoplasm (1,20,31). This activity is commonly studied in vitro with artificial liposomes filled with a fluorescent dye that is quenched within the liposome but fluoresces strongly when the liposome is breached and the dye is released (2,16). The role of the lytic peptide, and associated infectivity, in Flock House virus (FHV), a nonenveloped, Tϭ3 quasi-equivalent, small-subunit RNA (ssRNA) insect virus, was investigated with extensive mutagenesis in vivo employing a novel protocol in which the lytic peptide was provided in trans with a virus-like particle (VLP) (32).…”