scholarly journals Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

2011 ◽  
Vol 92 (12) ◽  
pp. 2838-2848 ◽  
Author(s):  
Shigeo Nagashima ◽  
Masaharu Takahashi ◽  
Suljid Jirintai ◽  
Toshinori Tanaka ◽  
Tsutomu Nishizawa ◽  
...  
Keyword(s):  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Hepatitis E ◽  
Negative Control ◽  
Dominant Negative ◽  
Wild Type ◽  
Orf3 Protein ◽  
Virus Particles ◽  
Virion Release ◽  
Vacuolar Protein

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 wild-type 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 wild-type 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.

Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies

2014 ◽  
Vol 95 (10) ◽  
pp. 2166-2175 ◽  
Author(s):  
Shigeo Nagashima ◽  
Suljid Jirintai ◽  
Masaharu Takahashi ◽  
Tominari Kobayashi ◽  
Tanggis ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Lipid Membrane ◽  
Hepatitis E ◽  
Negative Control ◽  
Bafilomycin A1 ◽  
Genotype 3 ◽  
Electron Microscopic ◽  
Immunogold Staining ◽  
Virus Particles ◽  
Virion Release

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

scholarly journals ORF3 protein of hepatitis E virus is essential for virion release from infected cells

2009 ◽  
Vol 90 (8) ◽  
pp. 1880-1891 ◽  
Author(s):  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Yu Hoshino ◽  
Hideyuki Takahashi ◽  
Koji Ichiyama ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Culture Medium ◽  
Culture Supernatant ◽  
Limit Of Detection ◽  
A549 Cells ◽  
Hepatitis E ◽  
Wild Type ◽  
Orf3 Protein ◽  
Virion Release ◽  
Infected Cells

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (ΔORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The ΔORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of ΔORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of ΔORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the ΔORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15–1.16 g ml−1, in contrast with the ΔORF3 mutant in culture supernatant, which banded at 1.27–1.28 g ml−1, similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.

scholarly journals Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation

2009 ◽  
Vol 83 (20) ◽  
pp. 10797-10807 ◽  
Author(s):  
Ritesh Tandon ◽  
David P. AuCoin ◽  
Edward S. Mocarski
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Body Protein ◽  
Escrt Machinery ◽  
Hcmv Replication ◽  
Viral Maturation ◽  
Vacuolar Protein

ABSTRACT The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

scholarly journals Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins

2019 ◽  
Vol 116 (21) ◽  
pp. 10402-10411 ◽  
Author(s):  
Nydia Tejeda-Muñoz ◽  
Lauren V. Albrecht ◽  
Maggie H. Bui ◽  
Edward M. De Robertis
Keyword(s):  
Wnt Signaling ◽  
Cancer Progression ◽  
Glycogen Synthase ◽  
Extracellular Fluid ◽  
Multivesicular Body ◽  
Extracellular Proteins ◽  
Dominant Negative ◽  
Canonical Wnt Signaling ◽  
Vacuolar Protein ◽  
Canonical Wnt

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator β-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

scholarly journals HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway

2004 ◽  
Vol 168 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Juan Martin-Serrano ◽  
Scott W. Eastman ◽  
Wayne Chung ◽  
Paul D. Bieniasz
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Sorting ◽  
Ubiquitin Ligases ◽  
Dominant Negative ◽  
Specific Class ◽  
Vacuolar Protein Sorting ◽  
Viral Budding ◽  
Virion Release ◽  
Vacuolar Protein ◽  
Class E

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif–dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.

scholarly journals The Interferon-Inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion

2015 ◽  
Vol 89 (18) ◽  
pp. 9200-9212 ◽  
Author(s):  
Ran Yan ◽  
Xuesen Zhao ◽  
Dawei Cai ◽  
Yuanjie Liu ◽  
Timothy M. Block ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Gpi Anchor ◽  
Enveloped Viruses ◽  
Virion Release ◽  
B Virus ◽  
Subviral Particles

ABSTRACTInterferon alpha (IFN-α) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-α elicits a pleiotropic antiviral state in hepatitis B virus (HBV)-infected hepatocytes, but whether or not IFN-α impedes the late steps of the HBV life cycle, such as HBV secretion, remains elusive. Here we report that IFN-α treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon-stimulated gene(s) that is responsible for the reduction of HBV virion release, we found that tetherin, a broad-spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-α in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, GPI anchor-truncated tetherin exhibited a dominant-negative effect and was incorporated into the liberated virions. We also found colocalization of tetherin and HBV L protein at the intracellular multivesicular body, where the budding of HBV virions takes place. In line with this, electron microscopy demonstrated that HBV virions were tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knockdown of tetherin or overexpression of dominant negative tetherin attenuated the IFN-α-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-α inhibits HBV virion egress from hepatocytes through the induction of tetherin.IMPORTANCETetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface with its membrane anchor domains. Here we report that interferon directly and selectively inhibits the secretion of HBV virions, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to liberated HBV virions. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virions occurs in the intracellular cisterna and that tetherin colocalizes with HBV virions on the multivesicular body, which is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.

scholarly journals Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus

2008 ◽  
Vol 83 (5) ◽  
pp. 2327-2337 ◽  
Author(s):  
Larissa Kolesnikova ◽  
Thomas Strecker ◽  
Eiji Morita ◽  
Florian Zielecki ◽  
Eva Mittler ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Protein Sorting ◽  
Marburg Virus ◽  
Dominant Negative ◽  
Cellular Proteins ◽  
Progeny Virus ◽  
Vacuolar Protein Sorting ◽  
Hek 293 ◽  
Virus Particles ◽  
Vacuolar Protein

ABSTRACT VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism.

scholarly journals A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells

2010 ◽  
Vol 92 (2) ◽  
pp. 269-278 ◽  
Author(s):  
S. Nagashima ◽  
M. Takahashi ◽  
Jirintai ◽  
T. Tanaka ◽  
K. Yamada ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Orf3 Protein ◽  
Virion Release ◽  
Infected Cells

scholarly journals The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains.

1992 ◽  
Vol 119 (4) ◽  
pp. 773-786 ◽  
Author(s):  
C A Vater ◽  
C K Raymond ◽  
K Ekena ◽  
I Howald-Stevenson ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Dominant Negative ◽  
Mutant Form ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Amino Terminal ◽  
Gtp Binding ◽  
Vacuolar Protein ◽  
Carboxy Terminal

The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.

scholarly journals The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 651
Author(s):  
Hsiao-Cheng Tsai ◽  
Che-Hong Chen ◽  
Daria Mochly-Rosen ◽  
Yi-Chen Ethan Li ◽  
Min-Huey Chen
Keyword(s):  
Bone Loss ◽  
Bacterial Infection ◽  
Bone Regeneration ◽  
Alcohol Drinking ◽  
Bacterial Species ◽  
Dominant Negative ◽  
Enzyme Inactivation ◽  
Wild Type ◽  
Micro Computed Tomography ◽  
Dominant Negative Mutation

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

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