Interaction of Tsg101 with Marburg Virus VP40 Depends on the PPPY Motif, but Not the PT/SAP Motif as in the Case of Ebola Virus, and Tsg101 Plays a Critical Role in the Budding of Marburg Virus-Like Particles Induced by VP40, NP, and GP

Abstract

Marburg virus (MARV) VP40 is a matrix protein that can be released from mammalian cells in the form of virus-like particles (VLPs) and contains the PPPY sequence, which is an L-domain motif. Here, we demonstrate that the PPPY motif is important for VP40-induced VLP budding and that VLP production is significantly enhanced by coexpression of NP and GP. We show that Tsg101 interacts with VP40 depending on the presence of the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and plays an important role in VLP budding. These findings provide new insights into the mechanism of MARV budding.Marburg virus (MARV) is a member of the Filoviridae, a family of negative-sense RNA viruses which cause fatal hemorrhagic disease in both humans and nonhuman primates. At present, no approved vaccines or antiviral drugs are available to prevent and treat filoviral diseases.The RNA genome of MARV encodes seven polypeptides, including the glycoprotein (GP), the nucleoprotein (NP), RNA-dependent RNA polymerase (L), VP35, VP30, VP40, and VP24. VP40 is the most abundant virion matrix protein and plays a key role in virus assembly and budding (15,16,31). GP is the only surface protein of filoviruses and is assumed to be responsible for binding to cellular receptors and for fusion of the viral envelope with the cellular membrane in the course of viral entry into the cells (2). The nucleocapsid complex, which contains NP, VP35, L, and VP30, encapsulates the viral genome.Recent studies have indicated that viral matrix proteins play critical roles during the late stage of virus budding in many enveloped RNA viruses, including retro-, rhabdo-, filo-, arena-, and orthomyxoviruses, and when expressed alone in cells, they are released in the form of virus-like particles (VLPs). These viral proteins possess a so-called L-domain, containing PT/ SAP, PPXY, and YPXL, which are motifs critical for efficient budding (3, 4, 6-13, 21, 25, 27, 30, 34-36, 39). Most of the host factors that interact with the L domain are involved in the class E vacuolar protein-sorting pathway, suggesting that budding into the lumen of multivesicular bodies (MVBs) in late endosomes and viral budding at the plasma membrane are topologically identical and share a common mechanism. MARV VP40 protein is sufficient for the release of VLPs (15,16,31) and contains a PPXY motif near its N terminus (Fig. 1B), but the viral L domain and the cellular factors required for its budding have yet to be determined.To gain insight into the mechanism of MARV budding, we analyzed the function of the PPPY motif as an L domain as well as the cellular factors involved in VLP budding.The PPPY motif within VP40 is important for efficient VLP production. First, to confirm that the expression of MARV VP40 in cells can induce the budding of VLPs that are morphologically identical to virions, we constructed a VP40 expression vector for the wild type (WT), pMV-VP40, by insertion of the coding region of VP40, amplified by PCR using pTM-VP40 as a template, into the pCAAGS vector (16, 23...

Key Genomic Changes Necessary for an In Vivo Lethal Mouse Marburgvirus Variant Selection Process

“…Two identical amino acid changes occurred in VP40. One of the two mutations occurred in the PPPY motif (VP40:Y19H) that was proposed to mediate (41). The other VP40 mutation (VP40:D184N) was located at the interdomain connecting loop between the VP40 N-and C-terminal domains based on the crystal structure of EBOV VP40 (12,15).…”

Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus

“…Matrix proteins from many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and arenaviruses, are able to induce VLPs (10, 14, 18, 28-30, 48, 49, 52). Increasing evidence also indicates that budding activity, and thus the release of VLPs, is often influenced by a complex interplay with components of the endosomal sorting complexes required for transport (ESCRTs), which mainly constitute the vacuolar protein sorting (VPS) pathway (16,38,42,54). ESCRTs trigger the formation and budding of vesicles into the lumina of multivesicular bodies (MVBs), and the constituents of the ESCRTs are recycled by the activity of VPS4, an AAA-type ATPase.…”

Antibody targeting of TSG101 on influenza-infected cells

“…The broad usage of TSG101 by divergent influenza viruses is consistent with evidence that TSG101 is "hijacked" by many virus families, including flaviviruses, filoviruses and retroviruses. 9,12,18,22,23,[28][29][30][31][32][33][34][35][36][37][38] Such findings suggest a convergent process whereby different virus types have evolved to exploit a similar mechanism. This raises an interesting question for future investigation to ask if TSG101 antibody-based inhibition of influenza might be similarly applicable to these other virus types.…”