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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.

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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...

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa

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...

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wildtype ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wildtype Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles. INTRODUCTIONHepatitis E virus (HEV), a member of the genus Hepevirus in the family Hepeviridae, is the causative agent of acute or fulminant hepatitis E, which occurs in many parts of the world, principally as a water-borne infection in developing countries and zoonotically in industrialized countries (Chandra et al., 2008;Colson et al., 2010;Dalton et al., 2008;Purcell & Emerson, 2008;Tei et al., 2003;Yazaki et al., 2003). HEV is a non-enveloped small virus with a diameter of 27-32 nm, present in the bile and faeces of infected hosts, while HEV particles in the circulating blood and culture supernatant are found to be associated with lipids (Takahashi et al., 2008b(Takahashi et al., , 2010Yamada et al., 2009a). The HEV genome is a positive-sense, ssRNA composed of approximately 7200 nt, which is capped and polyadenylated (Kabrane-Lazizi et al., 1999;Tam et al., 1991). The genome consists of a 59 UTR, three ORFs, a 39 UTR and a poly(A) tail at the 39 terminus (Emerson & Purcell, 2007). ORF1 encodes non-structural proteins including a methyltransferase, papain-like cysteine protease, helicase and RNA-dependent RNA polymerase (Agrawal et al., 2001;Koonin et al., 1992). ORF2 and ORF3 overlap, and the ORF2 and ORF3 proteins are translated from a bicistronic subgenomic RNA 2.2 kb in length (Graff et al., 2006;Ichiyama et al., 2009). The ORF2 protein is the viral capsid protein of 660 a...

In this study, we have identified a novel Nedd4-like ubiquitin ligase, BUL1, as the host factor involved in budding of type D retrovirus Mason-Pfizer monkey virus (M-PMV). Overexpression of BUL1 enhanced virus particle release, while a BUL1 mutant in which a W to G substitution was introduced into a WW domain, W791G, lost the ability to bind to the viral Gag protein and abolished its ability to mediate virus budding. In addition, a fragment of BUL1 containing only the WW domains inhibited virus budding in a dominant negative manner. These results, together with previous findings, indicate that the M-PMV Gag L domain interacts with the BUL1 WW domain and that this interaction is essential for virus budding. Our observations provide new insights into the mechanism of virus budding, and could be useful in establishing new antiviral strategies targeted at progeny virus release from a host cell.