Recently, tetherin has been identified as an effective cellular factor that prevents the release of human immunodeficiency virus type 1. Here, we show that the production of virus-like particles induced by viral matrix proteins of Lassa virus or Marburg virus was markedly inhibited by tetherin and that N-linked glycosylation of tetherin was dispensable for this antiviral activity. Our data also suggest that viral matrix proteins or one or more components that originate from host cells are targets of tetherin but that viral surface glycoproteins are not. These results suggest that tetherin inhibits the release of a wide variety of enveloped viruses from host cells by a common mechanism.

It is known that Lassa virus Z protein is sufficient for the release of virus-like particles (VLPs) and that it has two L domains, PTAP and PPPY, in its C terminus. However, little is known about the cellular factor for Lassa virus budding. We examined which cellular factors are used in Lassa virus Z budding. We demonstrated that Lassa Z protein efficiently produces VLPs and uses cellular factors, Vps4A, Vps4B, and Tsg101, in budding, suggesting that Lassa virus budding uses the multivesicular body pathway functionally. Our data may provide a clue to develop an effective antiviral strategy for Lassa virus.Lassa virus belongs to the family Arenaviridae, which includes Lymphocytic choriomeningitis virus (LCMV), Guanarito virus, Junin virus, and Machupo virus. Lassa virus is the etiological agent of a hemorrhagic fever, Lassa fever, that is endemic in West Africa.Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome. Lassa virus has two genomic RNA segments designated L and S, with approximate sizes of 7.2 and 3.4 kb, respectively. Each RNA segment directs the synthesis of two proteins in opposite orientations, separated by an intergenic region. The S segment directs synthesis of the nucleoprotein and the two virion glycoproteins, GP1 and GP2, which are derived by posttranslational cleavage of a precursor polypeptide, GP-C. The oligomeric structures of GP1 and GP2 make up the spikes on the virion envelope and mediate virus interaction with the host cell surface receptor (2). The L segment codes for the virus RNA-dependent RNA polymerase (L) and a small RING finger protein (Z), which is a viral matrix protein.Recent studies have revealed that viral matrix proteins play critical roles during a late stage of virus budding in many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, and orthomyxoviruses; 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 critical motifs for efficient budding (6, 9, 10, 12,17,19,(26)(27)(28)31). Lassa virus Z protein is sufficient for the release of VLPs and contains PTAP and PPXY motifs near its carboxy terminus (18,24).The PTAP motif was first identified in human immunodeficiency virus (HIV) p6 gag and has been reported to bind Tsg101, which is a ubiquitin-conjugating E2 variant with a component of the vesicular protein-sorting machinery. The interaction between p6 gag and Tsg101 is required for HIV budding, and Tsg101 appears to facilitate this budding by linking the p6 late domain to the vacuolar protein-sorting (Vps) pathway (5). Recent reports have shown that targeting of Tsg101 by RNA interference causes a strong reduction in Z-mediated LCMV budding and that the Z protein is colocalized with Tsg101 (18). Another L-domain motif, PPXY, has also been determined to be the principal sequence that binds to the WW domain, consisting of 38 to 40 amino acids containing two widely spaced tryptophans. In fa...

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...